EP2681330A1 - Use of the olfactomedin-4 protein (olfm4) in colorectal cancer diagnosis - Google Patents
Use of the olfactomedin-4 protein (olfm4) in colorectal cancer diagnosisInfo
- Publication number
- EP2681330A1 EP2681330A1 EP11719644.4A EP11719644A EP2681330A1 EP 2681330 A1 EP2681330 A1 EP 2681330A1 EP 11719644 A EP11719644 A EP 11719644A EP 2681330 A1 EP2681330 A1 EP 2681330A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- olfm4
- protein
- chemotherapeutic agent
- olfactomedin
- expression level
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 102100026071 Olfactomedin-4 Human genes 0.000 title claims abstract description 160
- 208000001333 Colorectal Neoplasms Diseases 0.000 title claims abstract description 91
- 206010009944 Colon cancer Diseases 0.000 title claims abstract description 80
- 101710109505 Olfactomedin-4 Proteins 0.000 title claims description 55
- 238000003745 diagnosis Methods 0.000 title claims description 8
- 238000000034 method Methods 0.000 claims abstract description 68
- 239000002246 antineoplastic agent Substances 0.000 claims abstract description 50
- 229940127089 cytotoxic agent Drugs 0.000 claims abstract description 48
- 238000011282 treatment Methods 0.000 claims abstract description 44
- 206010069755 K-ras gene mutation Diseases 0.000 claims abstract description 24
- 101001120760 Homo sapiens Olfactomedin-4 Proteins 0.000 claims abstract description 12
- 108090000623 proteins and genes Proteins 0.000 claims description 108
- 230000014509 gene expression Effects 0.000 claims description 85
- 239000000523 sample Substances 0.000 claims description 72
- 238000002493 microarray Methods 0.000 claims description 30
- 239000012472 biological sample Substances 0.000 claims description 19
- 239000003795 chemical substances by application Substances 0.000 claims description 19
- 230000000973 chemotherapeutic effect Effects 0.000 claims description 16
- 229960001756 oxaliplatin Drugs 0.000 claims description 16
- DWAFYCQODLXJNR-BNTLRKBRSA-L oxaliplatin Chemical compound O1C(=O)C(=O)O[Pt]11N[C@@H]2CCCC[C@H]2N1 DWAFYCQODLXJNR-BNTLRKBRSA-L 0.000 claims description 16
- 239000003814 drug Substances 0.000 claims description 15
- 229940079593 drug Drugs 0.000 claims description 14
- 101100242089 Homo sapiens OLFM4 gene Proteins 0.000 claims description 12
- 101150026723 OLFM4 gene Proteins 0.000 claims description 12
- 210000004369 blood Anatomy 0.000 claims description 12
- 239000008280 blood Substances 0.000 claims description 12
- 108020004707 nucleic acids Proteins 0.000 claims description 11
- 102000039446 nucleic acids Human genes 0.000 claims description 11
- 150000007523 nucleic acids Chemical class 0.000 claims description 11
- 238000003753 real-time PCR Methods 0.000 claims description 11
- 210000004881 tumor cell Anatomy 0.000 claims description 11
- 108091034117 Oligonucleotide Proteins 0.000 claims description 10
- 231100000024 genotoxic Toxicity 0.000 claims description 10
- 230000001738 genotoxic effect Effects 0.000 claims description 10
- 206010061289 metastatic neoplasm Diseases 0.000 claims description 10
- 238000005516 engineering process Methods 0.000 claims description 9
- 230000001394 metastastic effect Effects 0.000 claims description 9
- 229960001972 panitumumab Drugs 0.000 claims description 9
- 239000003153 chemical reaction reagent Substances 0.000 claims description 8
- UWKQSNNFCGGAFS-XIFFEERXSA-N irinotecan Chemical compound C1=C2C(CC)=C3CN(C(C4=C([C@@](C(=O)OC4)(O)CC)C=4)=O)C=4C3=NC2=CC=C1OC(=O)N(CC1)CCC1N1CCCCC1 UWKQSNNFCGGAFS-XIFFEERXSA-N 0.000 claims description 8
- 230000028327 secretion Effects 0.000 claims description 8
- 229960005395 cetuximab Drugs 0.000 claims description 7
- 238000000338 in vitro Methods 0.000 claims description 7
- JYEFSHLLTQIXIO-SMNQTINBSA-N folfiri regimen Chemical compound FC1=CNC(=O)NC1=O.C1NC=2NC(N)=NC(=O)C=2N(C=O)C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1.C1=C2C(CC)=C3CN(C(C4=C([C@@](C(=O)OC4)(O)CC)C=4)=O)C=4C3=NC2=CC=C1OC(=O)N(CC1)CCC1N1CCCCC1 JYEFSHLLTQIXIO-SMNQTINBSA-N 0.000 claims description 6
- 229960004768 irinotecan Drugs 0.000 claims description 6
- 238000001514 detection method Methods 0.000 claims description 5
- 238000004393 prognosis Methods 0.000 claims description 5
- YXTKHLHCVFUPPT-YYFJYKOTSA-N (2s)-2-[[4-[(2-amino-5-formyl-4-oxo-1,6,7,8-tetrahydropteridin-6-yl)methylamino]benzoyl]amino]pentanedioic acid;(1r,2r)-1,2-dimethanidylcyclohexane;5-fluoro-1h-pyrimidine-2,4-dione;oxalic acid;platinum(2+) Chemical compound [Pt+2].OC(=O)C(O)=O.[CH2-][C@@H]1CCCC[C@H]1[CH2-].FC1=CNC(=O)NC1=O.C1NC=2NC(N)=NC(=O)C=2N(C=O)C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 YXTKHLHCVFUPPT-YYFJYKOTSA-N 0.000 claims description 4
- 229940082789 erbitux Drugs 0.000 claims description 4
- 239000012528 membrane Substances 0.000 claims description 4
- 210000002751 lymph Anatomy 0.000 claims description 3
- 210000002966 serum Anatomy 0.000 claims description 3
- 238000011287 therapeutic dose Methods 0.000 claims description 3
- 210000002700 urine Anatomy 0.000 claims description 2
- 230000004044 response Effects 0.000 abstract description 8
- 230000006978 adaptation Effects 0.000 abstract description 2
- 206010028980 Neoplasm Diseases 0.000 description 88
- 210000004027 cell Anatomy 0.000 description 75
- 102000004169 proteins and genes Human genes 0.000 description 74
- 235000018102 proteins Nutrition 0.000 description 67
- 210000001519 tissue Anatomy 0.000 description 54
- 102100030708 GTPase KRas Human genes 0.000 description 33
- 101000584612 Homo sapiens GTPase KRas Proteins 0.000 description 33
- 108090000765 processed proteins & peptides Proteins 0.000 description 30
- 238000010186 staining Methods 0.000 description 22
- 201000011510 cancer Diseases 0.000 description 21
- 108020004999 messenger RNA Proteins 0.000 description 21
- 238000004458 analytical method Methods 0.000 description 17
- 108020004635 Complementary DNA Proteins 0.000 description 16
- 230000002018 overexpression Effects 0.000 description 15
- 238000010804 cDNA synthesis Methods 0.000 description 14
- 239000002299 complementary DNA Substances 0.000 description 14
- 238000001262 western blot Methods 0.000 description 14
- 101100338491 Oryza sativa subsp. japonica HCT1 gene Proteins 0.000 description 13
- 101100495309 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) CDH1 gene Proteins 0.000 description 13
- 108020004414 DNA Proteins 0.000 description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 12
- 108700039887 Essential Genes Proteins 0.000 description 11
- 241000282414 Homo sapiens Species 0.000 description 11
- 208000029742 colonic neoplasm Diseases 0.000 description 11
- 102000004196 processed proteins & peptides Human genes 0.000 description 11
- 210000001072 colon Anatomy 0.000 description 10
- 238000003752 polymerase chain reaction Methods 0.000 description 10
- 102000001301 EGF receptor Human genes 0.000 description 9
- 108060006698 EGF receptor Proteins 0.000 description 9
- 230000003321 amplification Effects 0.000 description 9
- 238000009739 binding Methods 0.000 description 9
- 230000000875 corresponding effect Effects 0.000 description 9
- 238000003199 nucleic acid amplification method Methods 0.000 description 9
- 230000004083 survival effect Effects 0.000 description 9
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 8
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- 102000016914 ras Proteins Human genes 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 238000002512 chemotherapy Methods 0.000 description 7
- 230000035772 mutation Effects 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 238000001228 spectrum Methods 0.000 description 7
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 6
- 101150105104 Kras gene Proteins 0.000 description 6
- 108700020796 Oncogene Proteins 0.000 description 6
- 230000004913 activation Effects 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 238000002487 chromatin immunoprecipitation Methods 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 108010014186 ras Proteins Proteins 0.000 description 6
- 238000012163 sequencing technique Methods 0.000 description 6
- 238000004885 tandem mass spectrometry Methods 0.000 description 6
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 5
- 102100040505 HLA class II histocompatibility antigen, DR alpha chain Human genes 0.000 description 5
- 108010067802 HLA-DR alpha-Chains Proteins 0.000 description 5
- 241000283973 Oryctolagus cuniculus Species 0.000 description 5
- 101710189720 Porphobilinogen deaminase Proteins 0.000 description 5
- 102100034391 Porphobilinogen deaminase Human genes 0.000 description 5
- 101710170827 Porphobilinogen deaminase, chloroplastic Proteins 0.000 description 5
- 101710100896 Probable porphobilinogen deaminase Proteins 0.000 description 5
- 108010026552 Proteome Proteins 0.000 description 5
- 108091023040 Transcription factor Proteins 0.000 description 5
- 102000040945 Transcription factor Human genes 0.000 description 5
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 5
- 238000003556 assay Methods 0.000 description 5
- 239000000284 extract Substances 0.000 description 5
- 238000003364 immunohistochemistry Methods 0.000 description 5
- 108010048477 olfactomedin Proteins 0.000 description 5
- 230000001105 regulatory effect Effects 0.000 description 5
- 150000003384 small molecules Chemical class 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 102100040685 14-3-3 protein zeta/delta Human genes 0.000 description 4
- AQQSXKSWTNWXKR-UHFFFAOYSA-N 2-(2-phenylphenanthro[9,10-d]imidazol-3-yl)acetic acid Chemical compound C1(=CC=CC=C1)C1=NC2=C(N1CC(=O)O)C1=CC=CC=C1C=1C=CC=CC=12 AQQSXKSWTNWXKR-UHFFFAOYSA-N 0.000 description 4
- VVIAGPKUTFNRDU-UHFFFAOYSA-N 6S-folinic acid Natural products C1NC=2NC(N)=NC(=O)C=2N(C=O)C1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 VVIAGPKUTFNRDU-UHFFFAOYSA-N 0.000 description 4
- 102100027314 Beta-2-microglobulin Human genes 0.000 description 4
- 102100026031 Beta-glucuronidase Human genes 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- 102100021429 DNA-directed RNA polymerase II subunit RPB1 Human genes 0.000 description 4
- 102100021699 Eukaryotic translation initiation factor 3 subunit B Human genes 0.000 description 4
- 101710113436 GTPase KRas Proteins 0.000 description 4
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 4
- 101000964898 Homo sapiens 14-3-3 protein zeta/delta Proteins 0.000 description 4
- 101000756632 Homo sapiens Actin, cytoplasmic 1 Proteins 0.000 description 4
- 101000937544 Homo sapiens Beta-2-microglobulin Proteins 0.000 description 4
- 101000933465 Homo sapiens Beta-glucuronidase Proteins 0.000 description 4
- 101001106401 Homo sapiens DNA-directed RNA polymerase II subunit RPB1 Proteins 0.000 description 4
- 101000896557 Homo sapiens Eukaryotic translation initiation factor 3 subunit B Proteins 0.000 description 4
- 101000988834 Homo sapiens Hypoxanthine-guanine phosphoribosyltransferase Proteins 0.000 description 4
- 101001067833 Homo sapiens Peptidyl-prolyl cis-trans isomerase A Proteins 0.000 description 4
- 101000579123 Homo sapiens Phosphoglycerate kinase 1 Proteins 0.000 description 4
- 101000662049 Homo sapiens Polyubiquitin-C Proteins 0.000 description 4
- 101000835093 Homo sapiens Transferrin receptor protein 1 Proteins 0.000 description 4
- 206010027476 Metastases Diseases 0.000 description 4
- KJWZYMMLVHIVSU-IYCNHOCDSA-N PGK1 Chemical compound CCCCC[C@H](O)\C=C\[C@@H]1[C@@H](CCCCCCC(O)=O)C(=O)CC1=O KJWZYMMLVHIVSU-IYCNHOCDSA-N 0.000 description 4
- 102100034539 Peptidyl-prolyl cis-trans isomerase A Human genes 0.000 description 4
- 102100028251 Phosphoglycerate kinase 1 Human genes 0.000 description 4
- 102100037935 Polyubiquitin-C Human genes 0.000 description 4
- 101710156592 Putative TATA-binding protein pB263R Proteins 0.000 description 4
- XYONNSVDNIRXKZ-UHFFFAOYSA-N S-methyl methanethiosulfonate Chemical compound CSS(C)(=O)=O XYONNSVDNIRXKZ-UHFFFAOYSA-N 0.000 description 4
- 108020004459 Small interfering RNA Proteins 0.000 description 4
- 102100040296 TATA-box-binding protein Human genes 0.000 description 4
- 101710145783 TATA-box-binding protein Proteins 0.000 description 4
- 102100026144 Transferrin receptor protein 1 Human genes 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 230000002596 correlated effect Effects 0.000 description 4
- 229960003722 doxycycline Drugs 0.000 description 4
- XQTWDDCIUJNLTR-CVHRZJFOSA-N doxycycline monohydrate Chemical compound O.O=C1C2=C(O)C=CC=C2[C@H](C)[C@@H]2C1=C(O)[C@]1(O)C(=O)C(C(N)=O)=C(O)[C@@H](N(C)C)[C@@H]1[C@H]2O XQTWDDCIUJNLTR-CVHRZJFOSA-N 0.000 description 4
- VVIAGPKUTFNRDU-ABLWVSNPSA-N folinic acid Chemical compound C1NC=2NC(N)=NC(=O)C=2N(C=O)C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 VVIAGPKUTFNRDU-ABLWVSNPSA-N 0.000 description 4
- 235000008191 folinic acid Nutrition 0.000 description 4
- 239000011672 folinic acid Substances 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 4
- 230000006698 induction Effects 0.000 description 4
- 230000000977 initiatory effect Effects 0.000 description 4
- 150000002500 ions Chemical class 0.000 description 4
- 229960001691 leucovorin Drugs 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000000816 matrix-assisted laser desorption--ionisation Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000011002 quantification Methods 0.000 description 4
- 239000000377 silicon dioxide Substances 0.000 description 4
- 238000007619 statistical method Methods 0.000 description 4
- HNSDLXPSAYFUHK-UHFFFAOYSA-N 1,4-bis(2-ethylhexyl) sulfosuccinate Chemical compound CCCCC(CC)COC(=O)CC(S(O)(=O)=O)C(=O)OCC(CC)CCCC HNSDLXPSAYFUHK-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 102100025064 Cellular tumor antigen p53 Human genes 0.000 description 3
- 206010052358 Colorectal cancer metastatic Diseases 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 description 3
- XKMLYUALXHKNFT-UUOKFMHZSA-N Guanosine-5'-triphosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XKMLYUALXHKNFT-UUOKFMHZSA-N 0.000 description 3
- 101000721661 Homo sapiens Cellular tumor antigen p53 Proteins 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 102000043276 Oncogene Human genes 0.000 description 3
- 239000012980 RPMI-1640 medium Substances 0.000 description 3
- 229920004890 Triton X-100 Polymers 0.000 description 3
- 239000013504 Triton X-100 Substances 0.000 description 3
- 238000011226 adjuvant chemotherapy Methods 0.000 description 3
- 150000001413 amino acids Chemical group 0.000 description 3
- 239000011324 bead Substances 0.000 description 3
- 231100000504 carcinogenesis Toxicity 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000009643 clonogenic assay Methods 0.000 description 3
- 231100000096 clonogenic assay Toxicity 0.000 description 3
- 230000001419 dependent effect Effects 0.000 description 3
- 230000003828 downregulation Effects 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 238000002509 fluorescent in situ hybridization Methods 0.000 description 3
- 229960002949 fluorouracil Drugs 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 108091008147 housekeeping proteins Proteins 0.000 description 3
- 238000001114 immunoprecipitation Methods 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 238000001155 isoelectric focusing Methods 0.000 description 3
- 238000000370 laser capture micro-dissection Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000004949 mass spectrometry Methods 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- 238000000751 protein extraction Methods 0.000 description 3
- 238000012175 pyrosequencing Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- CXVGEDCSTKKODG-UHFFFAOYSA-N sulisobenzone Chemical compound C1=C(S(O)(=O)=O)C(OC)=CC(O)=C1C(=O)C1=CC=CC=C1 CXVGEDCSTKKODG-UHFFFAOYSA-N 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000001356 surgical procedure Methods 0.000 description 3
- 238000002626 targeted therapy Methods 0.000 description 3
- 238000010361 transduction Methods 0.000 description 3
- 230000026683 transduction Effects 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- 230000003827 upregulation Effects 0.000 description 3
- 238000010200 validation analysis Methods 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 102000010400 1-phosphatidylinositol-3-kinase activity proteins Human genes 0.000 description 2
- DHMYGZIEILLVNR-UHFFFAOYSA-N 5-fluoro-1-(oxolan-2-yl)pyrimidine-2,4-dione;1h-pyrimidine-2,4-dione Chemical compound O=C1C=CNC(=O)N1.O=C1NC(=O)C(F)=CN1C1OCCC1 DHMYGZIEILLVNR-UHFFFAOYSA-N 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- 241000945470 Arcturus Species 0.000 description 2
- 241000972773 Aulopiformes Species 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 208000005623 Carcinogenesis Diseases 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 2
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 2
- 102100031480 Dual specificity mitogen-activated protein kinase kinase 1 Human genes 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102400001368 Epidermal growth factor Human genes 0.000 description 2
- 101800003838 Epidermal growth factor Proteins 0.000 description 2
- 108050001049 Extracellular proteins Proteins 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 108090000288 Glycoproteins Proteins 0.000 description 2
- 102000003886 Glycoproteins Human genes 0.000 description 2
- 101150034920 Hmbs gene Proteins 0.000 description 2
- 108010068342 MAP Kinase Kinase 1 Proteins 0.000 description 2
- 102000043136 MAP kinase family Human genes 0.000 description 2
- 108091054455 MAP kinase family Proteins 0.000 description 2
- 108091006036 N-glycosylated proteins Proteins 0.000 description 2
- 239000000020 Nitrocellulose Substances 0.000 description 2
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 2
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 2
- 108091007960 PI3Ks Proteins 0.000 description 2
- 102000000447 Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase Human genes 0.000 description 2
- 108010055817 Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase Proteins 0.000 description 2
- 108091000080 Phosphotransferase Proteins 0.000 description 2
- 102000009572 RNA Polymerase II Human genes 0.000 description 2
- 108010009460 RNA Polymerase II Proteins 0.000 description 2
- 208000005718 Stomach Neoplasms Diseases 0.000 description 2
- PZBFGYYEXUXCOF-UHFFFAOYSA-N TCEP Chemical compound OC(=O)CCP(CCC(O)=O)CCC(O)=O PZBFGYYEXUXCOF-UHFFFAOYSA-N 0.000 description 2
- 102000004243 Tubulin Human genes 0.000 description 2
- 108090000704 Tubulin Proteins 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- 230000000692 anti-sense effect Effects 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 239000012298 atmosphere Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 208000036815 beta tubulin Diseases 0.000 description 2
- 238000001574 biopsy Methods 0.000 description 2
- 229930189065 blasticidin Natural products 0.000 description 2
- 230000036952 cancer formation Effects 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000024245 cell differentiation Effects 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 239000012829 chemotherapy agent Substances 0.000 description 2
- 229940044683 chemotherapy drug Drugs 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- JBIWCJUYHHGXTC-AKNGSSGZSA-N doxycycline Chemical compound O=C1C2=C(O)C=CC=C2[C@H](C)[C@@H]2C1=C(O)[C@]1(O)C(=O)C(C(N)=O)=C(O)[C@@H](N(C)C)[C@@H]1[C@H]2O JBIWCJUYHHGXTC-AKNGSSGZSA-N 0.000 description 2
- 229940116977 epidermal growth factor Drugs 0.000 description 2
- 210000000981 epithelium Anatomy 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000005194 fractionation Methods 0.000 description 2
- 206010017758 gastric cancer Diseases 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 230000013595 glycosylation Effects 0.000 description 2
- 238000006206 glycosylation reaction Methods 0.000 description 2
- QGWNDRXFNXRZMB-UHFFFAOYSA-N guanidine diphosphate Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(COP(O)(=O)OP(O)(O)=O)C(O)C1O QGWNDRXFNXRZMB-UHFFFAOYSA-N 0.000 description 2
- 238000002991 immunohistochemical analysis Methods 0.000 description 2
- 238000012744 immunostaining Methods 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 230000004807 localization Effects 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 238000002595 magnetic resonance imaging Methods 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 150000002739 metals Chemical class 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 238000012775 microarray technology Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 229920001220 nitrocellulos Polymers 0.000 description 2
- 239000002853 nucleic acid probe Substances 0.000 description 2
- 238000011275 oncology therapy Methods 0.000 description 2
- 239000012188 paraffin wax Substances 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 102000020233 phosphotransferase Human genes 0.000 description 2
- 229920003023 plastic Polymers 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 108700042226 ras Genes Proteins 0.000 description 2
- 230000007115 recruitment Effects 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 238000002271 resection Methods 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 230000004043 responsiveness Effects 0.000 description 2
- 235000019515 salmon Nutrition 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000002002 slurry Substances 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 201000011549 stomach cancer Diseases 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000013589 supplement Substances 0.000 description 2
- UMGDCJDMYOKAJW-UHFFFAOYSA-N thiourea Chemical compound NC(N)=S UMGDCJDMYOKAJW-UHFFFAOYSA-N 0.000 description 2
- 229950003937 tolonium Drugs 0.000 description 2
- HNONEKILPDHFOL-UHFFFAOYSA-M tolonium chloride Chemical compound [Cl-].C1=C(C)C(N)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 HNONEKILPDHFOL-UHFFFAOYSA-M 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 2
- 239000008096 xylene Substances 0.000 description 2
- ZROHGHOFXNOHSO-BNTLRKBRSA-N (1r,2r)-cyclohexane-1,2-diamine;oxalic acid;platinum(2+) Chemical compound [Pt+2].OC(=O)C(O)=O.N[C@@H]1CCCC[C@H]1N ZROHGHOFXNOHSO-BNTLRKBRSA-N 0.000 description 1
- FDKXTQMXEQVLRF-ZHACJKMWSA-N (E)-dacarbazine Chemical compound CN(C)\N=N\c1[nH]cnc1C(N)=O FDKXTQMXEQVLRF-ZHACJKMWSA-N 0.000 description 1
- UMCMPZBLKLEWAF-BCTGSCMUSA-N 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCC[N+](C)(C)CCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 UMCMPZBLKLEWAF-BCTGSCMUSA-N 0.000 description 1
- AOJJSUZBOXZQNB-VTZDEGQISA-N 4'-epidoxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-VTZDEGQISA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- JYCQQPHGFMYQCF-UHFFFAOYSA-N 4-tert-Octylphenol monoethoxylate Chemical compound CC(C)(C)CC(C)(C)C1=CC=C(OCCO)C=C1 JYCQQPHGFMYQCF-UHFFFAOYSA-N 0.000 description 1
- STQGQHZAVUOBTE-UHFFFAOYSA-N 7-Cyan-hept-2t-en-4,6-diinsaeure Natural products C1=2C(O)=C3C(=O)C=4C(OC)=CC=CC=4C(=O)C3=C(O)C=2CC(O)(C(C)=O)CC1OC1CC(N)C(O)C(C)O1 STQGQHZAVUOBTE-UHFFFAOYSA-N 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- ULXXDDBFHOBEHA-ONEGZZNKSA-N Afatinib Chemical compound N1=CN=C2C=C(OC3COCC3)C(NC(=O)/C=C/CN(C)C)=CC2=C1NC1=CC=C(F)C(Cl)=C1 ULXXDDBFHOBEHA-ONEGZZNKSA-N 0.000 description 1
- 102000010565 Apoptosis Regulatory Proteins Human genes 0.000 description 1
- 108010063104 Apoptosis Regulatory Proteins Proteins 0.000 description 1
- 108010039627 Aprotinin Proteins 0.000 description 1
- 108700009171 B-Cell Lymphoma 3 Proteins 0.000 description 1
- 102000052666 B-Cell Lymphoma 3 Human genes 0.000 description 1
- 101150072667 Bcl3 gene Proteins 0.000 description 1
- 241000283725 Bos Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- COVZYZSDYWQREU-UHFFFAOYSA-N Busulfan Chemical compound CS(=O)(=O)OCCCCOS(C)(=O)=O COVZYZSDYWQREU-UHFFFAOYSA-N 0.000 description 1
- GAGWJHPBXLXJQN-UORFTKCHSA-N Capecitabine Chemical compound C1=C(F)C(NC(=O)OCCCCC)=NC(=O)N1[C@H]1[C@H](O)[C@H](O)[C@@H](C)O1 GAGWJHPBXLXJQN-UORFTKCHSA-N 0.000 description 1
- GAGWJHPBXLXJQN-UHFFFAOYSA-N Capecitabine Natural products C1=C(F)C(NC(=O)OCCCCC)=NC(=O)N1C1C(O)C(O)C(C)O1 GAGWJHPBXLXJQN-UHFFFAOYSA-N 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- DLGOEMSEDOSKAD-UHFFFAOYSA-N Carmustine Chemical compound ClCCNC(=O)N(N=O)CCCl DLGOEMSEDOSKAD-UHFFFAOYSA-N 0.000 description 1
- 102000000844 Cell Surface Receptors Human genes 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 101710163595 Chaperone protein DnaK Proteins 0.000 description 1
- 108010077544 Chromatin Proteins 0.000 description 1
- 102000005636 Cyclic AMP Response Element-Binding Protein Human genes 0.000 description 1
- 108010045171 Cyclic AMP Response Element-Binding Protein Proteins 0.000 description 1
- NZNMSOFKMUBTKW-UHFFFAOYSA-N Cyclohexanecarboxylic acid Natural products OC(=O)C1CCCCC1 NZNMSOFKMUBTKW-UHFFFAOYSA-N 0.000 description 1
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 241000252212 Danio rerio Species 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- HTIJFSOGRVMCQR-UHFFFAOYSA-N Epirubicin Natural products COc1cccc2C(=O)c3c(O)c4CC(O)(CC(OC5CC(N)C(=O)C(C)O5)c4c(O)c3C(=O)c12)C(=O)CO HTIJFSOGRVMCQR-UHFFFAOYSA-N 0.000 description 1
- MPJKWIXIYCLVCU-UHFFFAOYSA-N Folinic acid Natural products NC1=NC2=C(N(C=O)C(CNc3ccc(cc3)C(=O)NC(CCC(=O)O)CC(=O)O)CN2)C(=O)N1 MPJKWIXIYCLVCU-UHFFFAOYSA-N 0.000 description 1
- QGWNDRXFNXRZMB-UUOKFMHZSA-N GDP Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O QGWNDRXFNXRZMB-UUOKFMHZSA-N 0.000 description 1
- 102100029974 GTPase HRas Human genes 0.000 description 1
- 102100039788 GTPase NRas Human genes 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 101710178376 Heat shock 70 kDa protein Proteins 0.000 description 1
- 101710152018 Heat shock cognate 70 kDa protein Proteins 0.000 description 1
- 101000584633 Homo sapiens GTPase HRas Proteins 0.000 description 1
- 101000744505 Homo sapiens GTPase NRas Proteins 0.000 description 1
- 101000746367 Homo sapiens Granulocyte colony-stimulating factor Proteins 0.000 description 1
- 101001086545 Homo sapiens Olfactomedin-like protein 1 Proteins 0.000 description 1
- 101000605639 Homo sapiens Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform Proteins 0.000 description 1
- 101000984753 Homo sapiens Serine/threonine-protein kinase B-raf Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 108090000144 Human Proteins Proteins 0.000 description 1
- 102000003839 Human Proteins Human genes 0.000 description 1
- XDXDZDZNSLXDNA-TZNDIEGXSA-N Idarubicin Chemical compound C1[C@H](N)[C@H](O)[C@H](C)O[C@H]1O[C@@H]1C2=C(O)C(C(=O)C3=CC=CC=C3C3=O)=C3C(O)=C2C[C@@](O)(C(C)=O)C1 XDXDZDZNSLXDNA-TZNDIEGXSA-N 0.000 description 1
- XDXDZDZNSLXDNA-UHFFFAOYSA-N Idarubicin Natural products C1C(N)C(O)C(C)OC1OC1C2=C(O)C(C(=O)C3=CC=CC=C3C3=O)=C3C(O)=C2CC(O)(C(C)=O)C1 XDXDZDZNSLXDNA-UHFFFAOYSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 208000022559 Inflammatory bowel disease Diseases 0.000 description 1
- 239000005411 L01XE02 - Gefitinib Substances 0.000 description 1
- 239000005551 L01XE03 - Erlotinib Substances 0.000 description 1
- 239000002136 L01XE07 - Lapatinib Substances 0.000 description 1
- 239000002118 L01XE12 - Vandetanib Substances 0.000 description 1
- GDBQQVLCIARPGH-UHFFFAOYSA-N Leupeptin Natural products CC(C)CC(NC(C)=O)C(=O)NC(CC(C)C)C(=O)NC(C=O)CCCN=C(N)N GDBQQVLCIARPGH-UHFFFAOYSA-N 0.000 description 1
- 239000012097 Lipofectamine 2000 Substances 0.000 description 1
- GQYIWUVLTXOXAJ-UHFFFAOYSA-N Lomustine Chemical compound ClCCN(N=O)C(=O)NC1CCCCC1 GQYIWUVLTXOXAJ-UHFFFAOYSA-N 0.000 description 1
- 206010027457 Metastases to liver Diseases 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000204031 Mycoplasma Species 0.000 description 1
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 description 1
- 230000004988 N-glycosylation Effects 0.000 description 1
- 102100032751 Olfactomedin-like protein 1 Human genes 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 241000282577 Pan troglodytes Species 0.000 description 1
- 241000009328 Perro Species 0.000 description 1
- 108090000430 Phosphatidylinositol 3-kinases Proteins 0.000 description 1
- 102000003993 Phosphatidylinositol 3-kinases Human genes 0.000 description 1
- 102100038332 Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform Human genes 0.000 description 1
- 229940079156 Proteasome inhibitor Drugs 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 241000713810 Rat sarcoma virus Species 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 101100221606 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) COS7 gene Proteins 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 102100027103 Serine/threonine-protein kinase B-raf Human genes 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- BPEGJWRSRHCHSN-UHFFFAOYSA-N Temozolomide Chemical compound O=C1N(C)N=NC2=C(C(N)=O)N=CN21 BPEGJWRSRHCHSN-UHFFFAOYSA-N 0.000 description 1
- 208000034841 Thrombotic Microangiopathies Diseases 0.000 description 1
- 108700009124 Transcription Initiation Site Proteins 0.000 description 1
- 206010066901 Treatment failure Diseases 0.000 description 1
- 239000007984 Tris EDTA buffer Substances 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 229960002833 aflibercept Drugs 0.000 description 1
- 108010081667 aflibercept Proteins 0.000 description 1
- 229940008201 allegra Drugs 0.000 description 1
- AFVLVVWMAFSXCK-VMPITWQZSA-N alpha-cyano-4-hydroxycinnamic acid Chemical compound OC(=O)C(\C#N)=C\C1=CC=C(O)C=C1 AFVLVVWMAFSXCK-VMPITWQZSA-N 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 238000004082 amperometric method Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000000340 anti-metabolite Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 229940100197 antimetabolite Drugs 0.000 description 1
- 239000002256 antimetabolite Substances 0.000 description 1
- 239000000074 antisense oligonucleotide Substances 0.000 description 1
- 238000012230 antisense oligonucleotides Methods 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 229960004405 aprotinin Drugs 0.000 description 1
- 238000004630 atomic force microscopy Methods 0.000 description 1
- 229940120638 avastin Drugs 0.000 description 1
- 229960002707 bendamustine Drugs 0.000 description 1
- YTKUWDBFDASYHO-UHFFFAOYSA-N bendamustine Chemical compound ClCCN(CCCl)C1=CC=C2N(C)C(CCCC(O)=O)=NC2=C1 YTKUWDBFDASYHO-UHFFFAOYSA-N 0.000 description 1
- 239000003124 biologic agent Substances 0.000 description 1
- 238000000225 bioluminescence resonance energy transfer Methods 0.000 description 1
- 239000000091 biomarker candidate Substances 0.000 description 1
- 230000006287 biotinylation Effects 0.000 description 1
- 238000007413 biotinylation Methods 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 229960001467 bortezomib Drugs 0.000 description 1
- GXJABQQUPOEUTA-RDJZCZTQSA-N bortezomib Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)B(O)O)NC(=O)C=1N=CC=NC=1)C1=CC=CC=C1 GXJABQQUPOEUTA-RDJZCZTQSA-N 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 229960002092 busulfan Drugs 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 230000005907 cancer growth Effects 0.000 description 1
- 230000005773 cancer-related death Effects 0.000 description 1
- 229960004117 capecitabine Drugs 0.000 description 1
- 229960004562 carboplatin Drugs 0.000 description 1
- 190000008236 carboplatin Chemical compound 0.000 description 1
- 229960005243 carmustine Drugs 0.000 description 1
- 230000006369 cell cycle progression Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 229960004630 chlorambucil Drugs 0.000 description 1
- JCKYGMPEJWAADB-UHFFFAOYSA-N chlorambucil Chemical compound OC(=O)CCCC1=CC=C(N(CCCl)CCCl)C=C1 JCKYGMPEJWAADB-UHFFFAOYSA-N 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 229960004316 cisplatin Drugs 0.000 description 1
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000003021 clonogenic effect Effects 0.000 description 1
- 201000010897 colon adenocarcinoma Diseases 0.000 description 1
- 230000004736 colon carcinogenesis Effects 0.000 description 1
- 230000000112 colonic effect Effects 0.000 description 1
- 230000001010 compromised effect Effects 0.000 description 1
- 238000002591 computed tomography Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 229960004397 cyclophosphamide Drugs 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000007711 cytoplasmic localization Effects 0.000 description 1
- 210000003674 cytoplasmic vesicle Anatomy 0.000 description 1
- 210000004292 cytoskeleton Anatomy 0.000 description 1
- 210000000172 cytosol Anatomy 0.000 description 1
- 229960003901 dacarbazine Drugs 0.000 description 1
- 229960000975 daunorubicin Drugs 0.000 description 1
- STQGQHZAVUOBTE-VGBVRHCVSA-N daunorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(C)=O)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 STQGQHZAVUOBTE-VGBVRHCVSA-N 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 231100000517 death Toxicity 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000022811 deglycosylation Effects 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 239000013578 denaturing buffer Substances 0.000 description 1
- 229940009976 deoxycholate Drugs 0.000 description 1
- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000013024 dilution buffer Substances 0.000 description 1
- 230000003292 diminished effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000007783 downstream signaling Effects 0.000 description 1
- 229960004679 doxorubicin Drugs 0.000 description 1
- 238000002848 electrochemical method Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000000572 ellipsometry Methods 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 238000003114 enzyme-linked immunosorbent spot assay Methods 0.000 description 1
- 229960001904 epirubicin Drugs 0.000 description 1
- 229960001433 erlotinib Drugs 0.000 description 1
- AAKJLRGGTJKAMG-UHFFFAOYSA-N erlotinib Chemical compound C=12C=C(OCCOC)C(OCCOC)=CC2=NC=NC=1NC1=CC=CC(C#C)=C1 AAKJLRGGTJKAMG-UHFFFAOYSA-N 0.000 description 1
- 229960005420 etoposide Drugs 0.000 description 1
- VJJPUSNTGOMMGY-MRVIYFEKSA-N etoposide Chemical compound COC1=C(O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H]4O[C@H](C)OC[C@H]4O3)O)[C@@H]3[C@@H]2C(OC3)=O)=C1 VJJPUSNTGOMMGY-MRVIYFEKSA-N 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 239000002350 fibrinopeptide Substances 0.000 description 1
- 238000011354 first-line chemotherapy Methods 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 238000002866 fluorescence resonance energy transfer Methods 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 229960002584 gefitinib Drugs 0.000 description 1
- XGALLCVXEZPNRQ-UHFFFAOYSA-N gefitinib Chemical compound C=12C=C(OCCCN3CCOCC3)C(OC)=CC2=NC=NC=1NC1=CC=C(F)C(Cl)=C1 XGALLCVXEZPNRQ-UHFFFAOYSA-N 0.000 description 1
- 238000003197 gene knockdown Methods 0.000 description 1
- 102000034356 gene-regulatory proteins Human genes 0.000 description 1
- 108091006104 gene-regulatory proteins Proteins 0.000 description 1
- 238000012252 genetic analysis Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000013412 genome amplification Methods 0.000 description 1
- 238000003205 genotyping method Methods 0.000 description 1
- 238000009650 gentamicin protection assay Methods 0.000 description 1
- 108091005608 glycosylated proteins Proteins 0.000 description 1
- 102000035122 glycosylated proteins Human genes 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000005802 health problem Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 229960000908 idarubicin Drugs 0.000 description 1
- 229960001101 ifosfamide Drugs 0.000 description 1
- HOMGKSMUEGBAAB-UHFFFAOYSA-N ifosfamide Chemical compound ClCCNP1(=O)OCCCN1CCCl HOMGKSMUEGBAAB-UHFFFAOYSA-N 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000002055 immunohistochemical effect Effects 0.000 description 1
- 239000012133 immunoprecipitate Substances 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- ZPNFWUPYTFPOJU-LPYSRVMUSA-N iniprol Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@H]2CSSC[C@H]3C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC=4C=CC=CC=4)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=4C=CC=CC=4)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC2=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]2N(CCC2)C(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N2[C@@H](CCC2)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N2[C@@H](CCC2)C(=O)N3)C(=O)NCC(=O)NCC(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](C(=O)N1)C(C)C)[C@@H](C)O)[C@@H](C)CC)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZPNFWUPYTFPOJU-LPYSRVMUSA-N 0.000 description 1
- 238000011396 initial chemotherapy Methods 0.000 description 1
- 238000005305 interferometry Methods 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 229960004891 lapatinib Drugs 0.000 description 1
- BCFGMOOMADDAQU-UHFFFAOYSA-N lapatinib Chemical compound O1C(CNCCS(=O)(=O)C)=CC=C1C1=CC=C(N=CN=C2NC=3C=C(Cl)C(OCC=4C=C(F)C=CC=4)=CC=3)C2=C1 BCFGMOOMADDAQU-UHFFFAOYSA-N 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- GDBQQVLCIARPGH-ULQDDVLXSA-N leupeptin Chemical compound CC(C)C[C@H](NC(C)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C=O)CCCN=C(N)N GDBQQVLCIARPGH-ULQDDVLXSA-N 0.000 description 1
- 108010052968 leupeptin Proteins 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 229960002247 lomustine Drugs 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 238000001906 matrix-assisted laser desorption--ionisation mass spectrometry Methods 0.000 description 1
- 238000000074 matrix-assisted laser desorption--ionisation tandem time-of-flight detection Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 229960004961 mechlorethamine Drugs 0.000 description 1
- HAWPXGHAZFHHAD-UHFFFAOYSA-N mechlorethamine Chemical compound ClCCN(C)CCCl HAWPXGHAZFHHAD-UHFFFAOYSA-N 0.000 description 1
- 229960001924 melphalan Drugs 0.000 description 1
- SGDBTWWWUNNDEQ-LBPRGKRZSA-N melphalan Chemical compound OC(=O)[C@@H](N)CC1=CC=C(N(CCCl)CCCl)C=C1 SGDBTWWWUNNDEQ-LBPRGKRZSA-N 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 230000008883 metastatic behaviour Effects 0.000 description 1
- 238000001531 micro-dissection Methods 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 229960001156 mitoxantrone Drugs 0.000 description 1
- KKZJGLLVHKMTCM-UHFFFAOYSA-N mitoxantrone Chemical compound O=C1C2=C(O)C=CC(O)=C2C(=O)C2=C1C(NCCNCCO)=CC=C2NCCNCCO KKZJGLLVHKMTCM-UHFFFAOYSA-N 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 210000000066 myeloid cell Anatomy 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000001613 neoplastic effect Effects 0.000 description 1
- 229950008835 neratinib Drugs 0.000 description 1
- ZNHPZUKZSNBOSQ-BQYQJAHWSA-N neratinib Chemical compound C=12C=C(NC\C=C\CN(C)C)C(OCC)=CC2=NC=C(C#N)C=1NC(C=C1Cl)=CC=C1OCC1=CC=CC=N1 ZNHPZUKZSNBOSQ-BQYQJAHWSA-N 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 229960000435 oblimersen Drugs 0.000 description 1
- MIMNFCVQODTQDP-NDLVEFNKSA-N oblimersen Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(S)(=O)O[C@@H]2[C@H](O[C@H](C2)N2C3=NC=NC(N)=C3N=C2)COP(O)(=S)O[C@@H]2[C@H](O[C@H](C2)N2C(N=C(N)C=C2)=O)COP(O)(=S)O[C@@H]2[C@H](O[C@H](C2)N2C(N=C(N)C=C2)=O)COP(O)(=S)O[C@@H]2[C@H](O[C@H](C2)N2C3=C(C(NC(N)=N3)=O)N=C2)COP(O)(=S)O[C@@H]2[C@H](O[C@H](C2)N2C(N=C(N)C=C2)=O)COP(O)(=S)O[C@@H]2[C@H](O[C@H](C2)N2C3=C(C(NC(N)=N3)=O)N=C2)COP(O)(=S)O[C@@H]2[C@H](O[C@H](C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=S)O[C@@H]2[C@H](O[C@H](C2)N2C3=C(C(NC(N)=N3)=O)N=C2)COP(O)(=S)O[C@@H]2[C@H](O[C@H](C2)N2C(N=C(N)C=C2)=O)COP(O)(=S)O[C@@H]2[C@H](O[C@H](C2)N2C3=C(C(NC(N)=N3)=O)N=C2)COP(O)(=S)O[C@@H]2[C@H](O[C@H](C2)N2C3=NC=NC(N)=C3N=C2)COP(O)(=S)O[C@@H]2[C@H](O[C@H](C2)N2C(N=C(N)C=C2)=O)COP(O)(=S)O[C@@H]2[C@H](O[C@H](C2)N2C(N=C(N)C=C2)=O)COP(O)(=S)O[C@@H]2[C@H](O[C@H](C2)N2C(N=C(N)C=C2)=O)COP(O)(=S)O[C@@H]2[C@H](O[C@H](C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=S)O[C@@H]2[C@H](O[C@H](C2)N2C(N=C(N)C=C2)=O)COP(O)(=S)O[C@@H]2[C@H](O[C@H](C2)N2C(NC(=O)C(C)=C2)=O)CO)[C@@H](O)C1 MIMNFCVQODTQDP-NDLVEFNKSA-N 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000002985 plastic film Substances 0.000 description 1
- 229920006255 plastic film Polymers 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 150000003057 platinum Chemical class 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 229940002612 prodrug Drugs 0.000 description 1
- 239000000651 prodrug Substances 0.000 description 1
- 210000002307 prostate Anatomy 0.000 description 1
- 239000003207 proteasome inhibitor Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 108010077182 raf Kinases Proteins 0.000 description 1
- 102000009929 raf Kinases Human genes 0.000 description 1
- 230000025915 regulation of apoptotic process Effects 0.000 description 1
- 230000008261 resistance mechanism Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 239000012487 rinsing solution Substances 0.000 description 1
- 108091008601 sVEGFR Proteins 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 239000004055 small Interfering RNA Substances 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 238000003153 stable transfection Methods 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 1
- 229960004964 temozolomide Drugs 0.000 description 1
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 229960000303 topotecan Drugs 0.000 description 1
- UCFGDBYHRUNTLO-QHCPKHFHSA-N topotecan Chemical compound C1=C(O)C(CN(C)C)=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 UCFGDBYHRUNTLO-QHCPKHFHSA-N 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 238000002834 transmittance Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 239000000107 tumor biomarker Substances 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 229960000241 vandetanib Drugs 0.000 description 1
- UHTHHESEBZOYNR-UHFFFAOYSA-N vandetanib Chemical compound COC1=CC(C(/N=CN2)=N/C=3C(=CC(Br)=CC=3)F)=C2C=C1OCC1CCN(C)CC1 UHTHHESEBZOYNR-UHFFFAOYSA-N 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 238000004832 voltammetry Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 239000012130 whole-cell lysate Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/28—Compounds containing heavy metals
- A61K31/282—Platinum compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/243—Platinum; Compounds thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57419—Specifically defined cancers of colon
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/118—Prognosis of disease development
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
- G01N2001/2833—Collecting samples on a sticky, tacky, adhesive surface
- G01N2001/284—Collecting samples on a sticky, tacky, adhesive surface using local activation of adhesive, i.e. Laser Capture Microdissection
Definitions
- colorectal cancer is the third most common form of cancer and the second leading cause of cancer-related death in the Western world. Management of colorectal cancer patients and selection of the best treatment protocol is thus a crucial health problem. The selection of the appropriate treatment is crucial for the patient, as well as for health economics reasons.
- colorectal cancer therapy The main options for colorectal cancer therapy are surgery and or chemotherapy, depending on the individual patient's tumor staging and other age and complexmibidity factors.
- Tumor resistance to treatment most often implies transduction signal or antiapoptotic proteins overexpression or mutation.
- KRAS is a member of the rat sarcoma virus (ras) gene family of oncogenes (including KRAS, HRAS, and NRAS) and encodes the guanosine diphosphate (GDP)- and guanosine triphosphate (GTP)-binding protein RAS that acts as a major intracellular signal transducer.
- ras rat sarcoma virus
- GDP guanosine diphosphate
- GTP guanosine triphosphate
- RAS recruits the oncogene RAF, which phosphorylates MAP2K-1 (mitogen-activated protein kinase kinase-1 ) and MAP2K-2, thus initiating MAPK (mitogen-activated protein kinase) signaling that ultimately leads to expression of proteins playing important roles in cell growth, differentiation, and survival.
- MAPK mitogen-activated protein kinase
- KRAS is one of the commonly mutated oncogenes in human cancers.
- KRAS mutations are found in 30-40% of tumors and represent together with APC one of the somatic alteration involved in the initiation of colorectal cancer. This mutation occurs early in the process of carcinogenesis, and is maintained at the various stages of disease progression, such as node involvement and metastatic spread.
- a recent study involving a large number of patients has demonstrated that mutated KRAS is associated with worse outcome in colorectal cancer progression, with effects being more pronounced in stage II and III disease (Nash, et al., Ann. Surg. Oncol., 17: 416- 424, 2010). The same group has shown, in another study (Nash, et al., Ann.
- KRAS mutation is associated with more rapid and aggressive metastatic behavior of colorectal liver metastases.
- KRAS mutation has been reported to induce drug resistance and treatment failure to epidermal-growth factor receptor (EGFR)-targeting therapeutics in metastatic colorectal cancer.
- EGFR epidermal-growth factor receptor
- KRAS mutations confer resistance to both cetuximab (Erbitux®) and panitumumab (Vectibix®) (Allegra et al., J. Clin. Oncol., 27: 2091 -2096, 2008; Linardou et al., Lancet Oncol., 9: 962-972, 2008). Consequently, KRAS genotyping is required in metastatic colorectal cancer before selecting a targeted therapy and wild type Kras gene is mandatory before anti EGFR drugs administration.
- Kras is actually a cornerstone for tumor resistance. Not only are KRAS mutations responsible for resistance to both cetuximab and panitumumab, they have been recently shown to confer colorectal tumor resistance to a platinum derivative agent, oxaliplatin (Richman et al., J Clin Oncol, 27(35): 5866-5867, 2009). Moreover, if adding anti EGFR-targeting drug to irinotecan has no effect, either positive or negative, in the treatment of KRAS mutated tumors, adding the said drug to oxaliplatin leads to deleterious effects (Douillard et al., J Clin Oncol, 28(31 ): 4697-4705 2010).
- olfactomedin 4 (OLFM4) is associated with a higher risk of carrying a KRAS mutation. Moreover, olfactomedin 4 overexpression is linked to chemotherapy treatment resistance.
- OLFM4 is a member of olfactomedin domain-containing protein family that has a relatively diverse coil-coil domain in the amino terminus and a well-conserved olfactomedin domain in the carboxy-terminus.
- the olfactomedin 4 protein is a secreted N-glycosylated protein, which can be assayed in the blood of patients.
- the OLFM4 gene is highly expressed in myeloid differentiation in active inflammatory bowel disease, and in certain cancers suggesting that OLFM4 might play an important role in cellular differentiation, inflammation, and cancer evolution (Zheng et al, Blood, 103: 1883-1890, 2004; Liu et al, Exp Cell Res., 312(10): 1785-1797, 2006).
- OLFM4 is involved in regulating cellular apoptosis and the proliferation of cancer cells (Zhang et al, Cancer Res., 64: 2474-2481 , 2004; Kobayashi et al, Cancer Sci., 98(3): 334-340, 2007).
- OLFM4 is overexpressed in colon cancer (Koshida et al., Cancer Sci., 98(3): 315-320, 2007) and the concentration of circulating olfactomedin 4 is higher in the blood of colorectal cancer patients than in the blood of control healthy people (Yasui et al., Int J Cancer., 125(10): 2383-2392, 2009).
- the present invention thus provides a method for diagnosing the presence of a KRAS mutation in a patient's colorectal cancer.
- elevated expression levels of the OLFM4 gene and/or olfactomedin 4 protein indicate the presence of the said KRAS mutation.
- the colorectal cancer is a metastatic colorectal tumor.
- the invention relates to a method for determining the presence of a KRAS mutation in a metastatic colorectal cancer, said method comprising the steps of: (a) determining from a biological sample of a metastatic colorectal tumor-suffering subject the expression level of OLFM4,
- olfactomedin 4" it is herein referred to a secreted, N-glycosylated protein of 510 residues, said protein comprising an olfactomedin domain.
- the said protein is a human protein.
- the said protein has an amino acid sequence as in NP_006409.3, and the OLFM4 gene, which encodes the said protein, has a nucleotide sequence as in NM_006418.3.
- the OLFM4 gene is conserved in chimpanzee, dog, cow, mouse, rat, chicken, and zebrafish; should the need arise, the person skilled in the art would easily be able to identify the corresponding gene in any of these species on the basis of the sequence homology of the said gene with the above-mentioned human gene.
- the present inventors have shown that the presence of a KRAS mutation in a colorectal cancer leads to overexpression of the OLFM4 gene in turn results in the overexpression of the olfactomedin 4 protein. It is thus possible to determine OLFM4 gene expression levels either by measuring the amount of the OLFM4 nucleic acid transcript (i.e. based on the OLFM4 mRNA content of the sample) or of the olfactomedin 4 protein (i.e. based on the olfactomedin 4 protein content of the sample).
- the KRAS-mutated colorectal tumors preferentially express the glycosylated form of the said olfactomedin 4.
- the expression level of OLFM4 can also be determined by measuring the glycosylated olfactomedin 4 protein levels, i.e. by measuring the amount of the glycosylated form of the olfactomedin 4 protein.
- the amount of the glycosylated form is compared to the level of the non glycosylated form, with a ratio of glycosylated versus non glycosylated forms of the olfactomedin 4 protein higher than 1 .4 indicating the presence of KRAS mutation in the colorectal cancer; preferentially, the said ratio is higher than 1 .5; more preferentially, it is higher than 1 .6; even more preferentially, it is higher than 1 .7.
- the method of the invention comprises the additional steps of measuring the amount of the glycosylated form of the olfactomedin 4 protein, measuring the amount of the non glycosylated form of the olfactomedin 4 protein, and calculating the ratio of glycosylated versus non glycosylated forms of the olfactomedin 4 protein.
- the said olfactomedin 4 protein is preferentially secreted, as evidenced by the preferential localization in secretion vesicles of the said olfactomedin 4 protein in KRAS-mutated tumor cells, as compared to non-mutated tumor cells or healthy tissue. It is thus also possible to detect the expression level of OLFM4 by measuring the amount of olfactomedin 4 protein. This can be achieved either by measuring directly the amount of secreted protein from a biological sample which does not contain any tumor cell, or, in an indirect way, by quantifying in the tumor cells the secretion vesicles which contain the said olfactomedin 4 protein.
- OLFM4 levels can be used to determine if a patient will respond or not to chemotherapy treatment.
- the present inventors have shown that overexpression of OLFM4 confers resistance to chemotherapy treatment.
- overexpression of OLFM4 increases the survival of colorectal cancer cells in the presence of genotoxic agents, such as oxaliplatin and sn38 (the active metabolite of irinotecan. More specifically, the presence of secreted olfactomedin 4 in the medium is sufficient to promote resistance of colon cancer cells to the said agents.
- genotoxic agents such as oxaliplatin and sn38
- the efficacy of an EGFR-targeting agent for treating colorectal cancer is dependent upon the absence of any KRAS mutation.
- the invention thus relates to a method for the in vitro diagnosis or prognosis of a chemotherapeutic agent-responding or non-responding phenotype, comprising:
- chemotherapeutic agent responding or non-responding phenotype from said comparison.
- “Chemotherapy” as used herein is a cancer treatment that uses drugs to stop the growth of cancer cells, either by killing the cells or by stopping them from dividing.
- the said drug can be for example a small molecule: small molecules which can be conveniently used for the invention include in particular genotoxic drugs.
- genotoxic drugs used for colorectal cancer treatment include busulfan, bendamustine, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, daunorubicin, doxorubicin, epirubicin, etoposide, idarubicin, ifosfamide, irinotecan (and its active metabolite sn38), lomustine, mechlorethamine, melphalan, mitomycin c, mitoxantrone, oxaliplatin, temozolamide and topotecan.
- the genotoxic drugs according to the invention are oxaliplatin, irinotecan, and irinotecan active metabolite sn38.
- the invention should not be understood as being limited to genotoxic drugs, as many other types of small molecules can also be used in the context of this invention.
- antimetabolites such as 5-FU (and its pro-drug capecitabine), tegafur-uracil (or UFT or UFUR), leucovorin (LV, folinic acid), or proteasome inhibitors such as bortezomib are also encompassed by the scope of this invention.
- a biological drug according to the invention is any type of biological agent which has a therapeutic activity in colorectal cancer therapy.
- the said agent can be for example an antisense oligonucleotide (e.g. oblimersen), but it is preferentially a protein such as a soluble VEGF receptor, e.g. aflibercept, or an antibody.
- the said agent is a monoclonal antibody (e.g. bevacuzimab, i.e. Avastin®).
- Another preferred group of chemotherapeutic drugs according to the invention corresponds to the EGFR-targeting agents.
- EGFR-targeting agent or "ETA”, it is herein meant an agent which is capable of neutralizing the effects of EGFR, i.e. an inhibitor of EGFR.
- EGFR epidermal growth factor family
- EGFR protein according to the invention is a human polypeptide having an aminoacid sequence as laid out in NP_005219.
- the said agent of the invention is capable of inhibiting the downstream signalling of EGFR.
- this class of agents is distinguished on the basis of its target, not by its nature (i.e. small molecule or biotherapeutic).
- ETAs include small molecules which bind to and inhibit EGFR, such as gefitinib, erlotinib, vandetanib, BIBW 2992, lapatinib, or neratinib.
- ETAs according to the invention also encompass antibodies, and in particular monoclonal antibodies.
- the said agent is selected in the group consisting of panitumumab (Vectibix®), mastuzumab, and cetuximab (Erbitux®). In an even more preferred embodiment, the said agent is cetuximab (Erbitux®).
- the invention also relates to a method for designing treatment with a chemotherapeutic agent for a subject suffering from a colorectal cancer, said method comprising:
- step (d) designing the dose of chemotherapeutic agent treatment according to said identified chemotherapeutic agent-responding or non-responding phenotype.
- the dose of chemotherapeutic treatment determined in step (b) may be equal to 0.
- the dose of chemotherapeutic agent determined in step (b) is administered to the subject. If the dose is equal to 0, then no treatment is given.
- the invention is also drawn to a method of treatment of a colorectal cancer-suffering subject with a chemotherapy agent, comprising:
- step (a) determining from a biological sample of the said colorectal cancer-suffering subject the presence of a chemotherapeutic agent-responding or non-responding phenotype using a method according to the invention, and (b) adapting the chemotherapeutic agent treatment in function of the result of step (a).
- Said adaptation of the chemotherapeutic agent treatment may consist in: a reduction or suppression of the said chemotherapeutic agent treatment if the subject has been diagnosed as chemotherapeutic agent non- responding, or - the continuation of the said treatment with said chemotherapeutic agent if the subject has been diagnosed as chemotherapeutic agent-responding.
- the invention also refers to a new use of a chemotherapeutic agent in the treatment of colorectal cancer, comprising the steps of:
- step (b) determining the dose of chemotherapeutic agent to administer with respect to the result of step (a).
- the dose of chemotherapeutic agent determined in step (b) is administered to the subject. As mentioned above, if the dose is equal to 0, then no treatment is given.
- the invention thus relates to a chemotherapeutic agent to treat colorectal cancer, wherein the chemotherapeutic agent is administered to a colorectal cancer-suffering subject who has been diagnosed and/or prognosed as responsive using a method according to the invention. More specifically, the invention relates to a chemotherapeutic agent to treat colorectal cancer in a subject suffering from a colorectal cancer, wherein: (a) the chemotherapeutic agent-responding or non-responding phenotype of the said subject is determined according to the method of the invention,
- step (c) the dose of the chemotherapeutic agent which is determined in step (b) is administered to the said subject.
- first line chemotherapy regimens for colorectal cancer involve the combination of infusional 5-fluorouracil, leucovorin, and oxaliplatin (FOLFOX) with cetuximab or panitumumab, or of infusional 5-fluorouracil, leucovorin, and irinotecan (FOLFIRI) with cetuximab or panitumumab, both in KRAS wild type tumors.
- FOLFOX infusional 5-fluorouracil, leucovorin, and oxaliplatin
- FOLFIRI infusional 5-fluorouracil, leucovorin, and irinotecan
- step (c) determining the dose of chemotherapeutic agent to administer with respect to the result of step (b).
- the invention also refers to a combination of chemotherapeutic agent and FOLFOX or chemotherapeutic agent and FOLFIRI for the treatment of colorectal cancer, comprising the steps of:
- step (c) determining the dose of chemotherapeutic agent to administer with respect to the result of step (b).
- the dose of chemotherapeutic agent determined in step (c) is administered to the subject.
- a "chemotherapeutic agent responding phenotype” is defined as a response state of a subject to the administration of a chemotherapeutic agent.
- a “response state” means that the said subject (referred to as a chemotherapeutic agent responding subject or a responding subject or a responsive subject: for the purpose of this application, these terms are similar) responds to the treatment, i.e. that the treatment is efficacious in the said subject.
- the definition of response is a reduction of tumor volume as assessed by e.g. CT-Scan or Magnetic resonance imaging (MRI). These criteria are thus well known to the skilled person in the art and need not be detailed here.
- chemotherapeutic agent non-responding phenotype refers to the absence in said subject (referred to herein as an chemotherapeutic agent non- responding subject or a non responding subject or a non-responsive subject: these terms should be construed in the context of this application as having the same meaning) of a state of response, meaning that said subject remains refractory to the treatment.
- the said subject is a colorectal cancer- suffering subject.
- a "colorectal cancer-suffering subject” is a subject with a cancer at any of the stages of the classification used by the person skilled in the art. In other words, any subject with a stage 0, a stage I, a stage 11 A, a stage I IB, a stage IIIA, a stage 1MB, a stage NIC, or a stage IV colorectal cancer, is a colorectal cancer-suffering subject as understood herein.
- the said subject is not treated with a chemotherapeutic agent; in another further embodiment, the said subject is treated with a chemotherapeutic agent.
- the methods of the invention permit a prognosis of the responsiveness/non responsiveness of the said subject.
- the method of the invention allows the person skilled in the art to prognose (i.e. to identify) the subjects susceptible of responding to the chemotherapeutic agent treatment. This is important because of the destructive and potentially fatal nature of colorectal cancer and the societal costs of inefficacious chemotherapeutic treatments.
- this embodiment of the invention allows for identification of non responsive subjects before any treatment is initiated, the risks for one treated subject to encounter severe adverse effects are greatly diminished.
- the methods of the invention are useful for diagnosing if a subject responds to the said chemotherapeutic agent, and whether the said subject would thus benefit from a continuation of the said treatment. Moreover, they are useful for diagnosing subjects who are not responding to the treatment, i.e. who are refractory to the chemotherapeutic agent, and should thus swiftly shifted to another therapy. In regard of the potentially lethal nature of colorectal cancer, this achievement is crucial.
- a “biological sample” may be any sample that may be taken from a subject, such as a serum sample, a plasma sample, a urine sample, a blood sample, a lymph sample, or a colorectal cancer sample. Such a sample must allow for the determination of the expression levels of OLFM4.
- Preferred biological samples for the determination of OLFM4 expression level by detection of the secreted olfactomedin 4 protein include samples such as a blood sample, a plasma sample, a lymph sample, or a colorectal cancer sample.
- the biological sample is a blood sample.
- a blood sample may be obtained by a completely harmless blood collection from the patient and thus allows for a non-invasive diagnosis of a chemotherapeutic agent responding or non-responding phenotype.
- a "biological sample” as used herein also includes a colorectal cancer sample of the patient to be tested. Such colorectal cancer sample allows the skilled person to perform any type of measurement of the level of OLFM4 and/or olfactomedin 4.
- the methods according to the invention may further comprise a preliminary step of taking a colon cancer sample from the patient.
- a colon cancer sample it is referred to a tumor colon tissue sample. Even in a cancer patient, colon tissue still comprises non tumor healthy tissue.
- the “colorectal cancer sample” should thus be limited to tumor colon tissue taken from the patient.
- Said “colorectal cancer sample” may be a biopsy sample or a sample taken from a surgical colon resection or a colorectal metastasis surgical resection.
- Colorectal cancer sample as used herein encompasses both colorectal primary tumors and colorectal metastatic tumors.
- the amount of nucleic acid transcripts can be measured by any technology known by a man skilled in the art. In particular, the measure may be carried out directly on an extracted messenger RNA (mRNA) sample, or on retrotranscribed complementary DNA (cDNA) prepared from extracted mRNA by technologies well known in the art. From the mRNA or cDNA sample, the amount of nucleic acid transcripts may be measured using any technology known by a person skilled in the art, including nucleic microarrays, quantitative PCR, and hybridization with a labelled probe.
- mRNA messenger RNA
- cDNA retrotranscribed complementary DNA
- the methods according to the invention may comprise another preliminary step, between the taking of the sample from the patient and steps a) as defined above, corresponding to the transformation of the colorectal cancer sample (and optionally of the healthy colon tissue sample) into a mRNA (or corresponding cDNA) sample or into a protein sample, which is then ready to use for in vitro measuring of genes expression levels in step a).
- Preparation or extraction of mRNA (as well as retrotranscription into cDNA) or proteins from a tissue sample is only routine procedure well known to those skilled in the art.
- the measure of OLFM4 gene expression levels may be performed, depending on the type of transformation and the available ready-to-use sample, either at the mRNA (i.e. based on the mRNA content of the sample) or at the protein level (i.e. based on the protein content of the sample).
- the expression levels of some of the genes may be measured at the mRNA level, while the expression levels of other genes are measured at the protein level.
- part of the colorectal cancer sample taken from the patient has been transformed into an mRNA (or corresponding cDNA) sample and another part has been transformed into a protein sample.
- the expression levels of all tested genes are measured either at the mRNA or at the protein level.
- the expression level is determined using quantitative PCR.
- Quantitative, or real-time, PCR is a well known and easily available technology for those skilled in the art and does not need a precise description.
- the determination of the expression level using quantitative PCR may be performed as follows. Briefly, the real-time PCR reactions are carried out using the TaqMan Universal PCR Master Mix (Applied Biosystems). 6 ⁇ _ cDNA is added to a 9 ⁇ _ PCR mixture containing 7.5 ⁇ _ TaqMan Universal PCR Master Mix, 0.75 ⁇ _ of a 20X mixture of probe and primers and 0.75 ⁇ _ water. The reaction consisted of one initiating step of 2 min at 50 deg. C, followed by 10 min at 95 deg. C, and 40 cycles of amplification including 15 sec at 95 deg. C and 1 min at 60 deg. C.
- the reaction and data acquisition can be performed using the ABI PRISM 7900 Sequence Detection System (Applied Biosystems).
- the number of template transcript molecules in a sample is determined by recording the amplification cycle in the exponential phase (cycle threshold or C T ), at which time the fluorescence signal can be detected above background fluorescence.
- the starting number of template transcript molecules is inversely related to C T .
- the expression level is determined by the use of a nucleic microarray.
- a "nucleic microarray” consists of different nucleic acid probes that are attached to a substrate, which can be a microchip, a glass slide or a microsphere-sized bead.
- a microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose.
- Probes can be nucleic acids such as cDNAs ("cDNA microarray”) or oligonucleotides (“oligonucleotide microarray”), and the oligonucleotides may be about 25 to about 60 base pairs or less in length.
- tissue microarrays coupled to fluorescent in situ hybridization may be used.
- Tissue microarrays also known as TMAs
- TMAs Tissue microarrays
- a hollow needle is used to remove tissue cores as small as 0.6 mm in diameter from regions of interest in paraffin-embedded tissues such as clinical biopsies or tumor samples. These tissue cores are then inserted in a recipient paraffin block in a precisely spaced, array pattern.
- Sections from this block are cut using a microtome, mounted on a microscope slide and then analyzed by any method of standard histological analysis.
- Each microarray block can be cut into 100 - 500 sections, which can be subjected to independent tests.
- Tests commonly employed in tissue microarray include immunohistochemistry, and fluorescent in situ hybridization.
- tissue microarray technology may be coupled to fluorescent in situ hybridization.
- expression levels When expression levels are measured at the protein level, it may be notably performed using specific antibodies, in particular using well known technologies such as cell membrane staining using biotinylation or other equivalent techniques followed by immunoprecipitation with specific antibodies, western blot, ELISA or ELISPOT, antibodies microarrays, or tissue microarrays coupled to immunohistochemistry.
- suitable techniques include FRET or BRET, single cell microscopic or histochemistery methods using single or multiple excitation wavelength and applying any of the adapted optical methods, such as electrochemical methods (voltametry and amperometry techniques), atomic force microscopy, and radio frequency methods, e.g.
- multipolar resonance spectroscopy confocal and non-confocal, detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, and birefringence or refractive index (e.g., surface plasmon resonance, ellipsometry, a resonant mirror method, a grating coupler waveguide method or interferometry), cell ELISA, flow cytometry, radioisotopic, magnetic resonance imaging, analysis by polyacrylamide gel electrophoresis (SDS-PAGE); HPLC-Mass Spectroscopy; Liquid Chromatography/Mass Spectrometry/Mass Spectrometry (LC- MS/MS)).
- fluorescence luminescence, chemiluminescence, absorbance, reflectance, transmittance, and birefringence or refractive index
- birefringence or refractive index e.g., surface plasmon resonance, ellipsometry, a resonant mirror method, a
- the glycosylated and the non glycosylated forms of the olfactomedin 4 protein can be assessed on the basis their respective different electrophoresis mobility by Western blotting with an antibody recognizing both forms.
- One example of such an assay is shown in the experimental examples.
- the total amount of olfactomedin 4 protein can be assessed by ELISA with an antibody recognizing both forms, and the fraction of glycosylated olfactomedin with an antibody specific for this form.
- the comparison of the expression levels of the measured genes in said patient's colorectal cancer sample is made by calculating an expression level ratio of the expression level of the OLFM4 gene to the expression level of a reference gene in said patient's colon cancer sample, and by comparing the obtained expression level ratio to a corresponding threshold value.
- Said reference gene is a gene which is expressed in all cell types. More specifically, the reference gene according to the invention is a gene which is expressed in all the cells constituting the colon. In another aspect, the expression level of the reference gene is not affected by the state of the cell, i.e. the control gene is expressed to the same level in a healthy cell and in a tumor cell. In a specific embodiment, the reference gene is a housekeeping gene.
- a housekeeping gene is a gene expressed in all cell types, which provides a basic function needed for sustenance of all cell types.
- a list of human housekeeping genes may be found in Eisenberg et al. (Trends in Genetics 19: 362-365, 2003).
- a preferred housekeeping gene according to the invention is a gene selected in the group consisting of B2M, TFRC, YWHAZ, RPLO, 18S, GUSB, UBC, TBP, GAPDH, PPIA, POLR2A, ACTB, PGK1 , HPRT1 , IP08 and HMBS.
- a "threshold value” is intended to mean a value that permits to discriminate samples in which the expression level ratio of the gene of interest corresponds to an expression level of said gene of interest in the patient's colon cancer sample that is low or high. In particular, if a gene expression level ratio is inferior or equal to the threshold value, then the expression level of this gene in the patient's colon cancer sample is considered low, whereas if a gene expression level ratio is superior to the threshold value, then the expression level of this gene in the patient's colon cancer sample is considered high. For each gene, and depending on the method used for measuring the expression level of the genes, the optimal threshold value may vary.
- control colorectal cancer samples in which the expression level (low or high) is known for this particular gene, and on the comparison thereof with the expression of a control gene, e.g. a housekeeping gene.
- the present invention further relates to a microarray dedicated to the implementation of the methods according to the invention, comprising at most 500, preferably at most 300, at most 200, more preferably at most 150, at most 100, even more preferably at most 75, at most 50, at most 40, at most 30, at most 20, at most 10 distinct probes, at least 1 of which specifically binds to OLFM4 mRNA (or corresponding cDNA) or protein.
- said microarray is a nucleic acid microarray, comprising at most 500, preferably at most 300, at most 200, more preferably at most 150, at most 100, even more preferably at most 75, at most 50, at most 40, at most 30, at most 20, at most 10 distinct probes (thus excluding for instance pangenomic microarrays), at least 1 of which specifically hybridizes to OLFM4 mRNA (or corresponding cDNA).
- Said microarray may also contain at least one probe which specifically hybridizes to a housekeeping gene in addition to the probe specifically hybridizing to OLFM4.
- said housekeeping gene is selected in the group consisting of B2M, TFRC, YWHAZ, RPLO, 18S, GUSB, UBC, TBP, GAPDH, PPIA, POLR2A, ACTB, PGK1 , HPRT1 , IP08 and HMBS. More preferentially, the housekeeping gene is the IP08 or the HMBS gene.
- a "nucleic microarray” consists of different nucleic acid probes that are attached to a substrate, which can be a microchip, a glass slide or a microsphere-sized bead.
- a microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose.
- Probes can be nucleic acids such as cDNAs ("cDNA microarray") or oligonucleotides ("oligonucleotide microarray", the oligonucleotides being about 25 to about 60 base pairs or less in length).
- said microarray may be a protein microarray.
- said microarray is an antibodies microarray, comprising at most 500, preferably at most 300, at most 200, more preferably at most 150, at most 100, even more preferably at most 75, at most 50, at most 40, at most 30, at most 20, at most 10 distinct antibodies, at least 1 of which specifically bind to olfactomedin 4 protein.
- Said microarray may also contain at least one antibody which specifically binds to a housekeeping protein, in addition to the antibody specifically binding to the olfactomedin 4 protein.
- said housekeeping protein is selected in the group consisting of the B2M, TFRC, YWHAZ, RPLO, 18S, GUSB, UBC, TBP, GAPDH, PPIA, POLR2A, ACTB, PGK1 , HPRT1 , IP08 and HMBS proteins.
- said housekeeping protein is the IP08 or HMBS protein.
- the present invention thus further relates to a kit for diagnosing the presence of a KRAS mutation in a colorectal cancer in a patient from a colorectal cancer sample of said patient, comprising at least one reagent for the determination of the OLFM4 expression level.
- the invention also relates to a kit for the in vitro diagnosis of a responding or non responding phenotype, comprising at least one reagent for the determination of the OLFM4 expression level.
- the kit of the invention comprises a dedicated microarray as described above or amplification primers specific for OLFM4.
- the kit comprises amplification primers
- said kit may comprise amplification primers specific for other genes
- said kit preferably comprises at most 100, at most 75, 50, at most 40, at most 30, preferably at most 25, at most 20, at most 15, more preferably at most 10, at most 8, at most 6, even more preferably at most 5, at most 4, at most 3 or even 2 or one or even zero couples of amplification primers specific for other genes than OLFM4.
- said kit may comprise at least a couple of amplification primers for at least one housekeeping gene in addition to the primers for OLFM4.
- said housekeeping gene is selected in the group consisting of B2M, TFRC, YWHAZ, RPLO, 18S, GUSB, UBC, TBP, GAPDH, PPIA, POLR2A, ACTB, PGK1 , HPRT1 , IP08 and HMBS.
- said housekeeping gene is the IP08 or HMBS gene.
- FIG. 1 OLFM4 is overexpressed in KRAS tumors.
- OLFM4 is glycosylated in KRAS tumors.
- N F-KB2 regulates OLFM4 expression following Ras induction.
- A. HT29-H-RasV12 colorectal cell lines were treated or not with doxycyclin for the indicated time, whole cell extracts were prepared and analyzed using antibodies directed against Ras, N F-KB2, OLFM4 and HSP70.
- B. Cells were treated as above and the mRNA expressions of OLFM4 and RPLPO were evaluated by semiquantitative RT-PCR.
- C Schematic representation of the potential N F- ⁇ binding sites of the OLFM4 promoter. The reading frame of the binding site is indicated by + for 5'- 3' and - for 3'-5'.
- HCT1 16 colorectal cell lines were either transfected with NF- KB2 specific or control siRNA oligonucleotides as indicated.
- the mRNA extracts (D) and whole cell extracts (E) were processed and the OLFM4 expression was analyzed 48hr after siRNA transfection.
- F. Soluble chromatin from growing HCT1 16 cells was prepared and immunoprecipitated with antibodies targeted against N F-KB2, BCI3 and the RNA polymerase II. DNA was amplified using pair of primers that cover the N F-KB proximal binding site of the OLFM4 promoter. ChIP assays were then quantified by real-time RT-PCR as compared to the signal obtained on a control sequence with a control IgG.
- OLFM4 The influence of OLFM4 following genotoxic treatments in HCT1 16 OLFM4 inducible cells was evaluated by clonogenic assay. Cells were incubated with 50 ng/ml of doxycyclin, and then 24hr later treated with oxaliplatin or sn38 at the indicated concentration for 10 days. The colonies were stained with crystal violet and counted by QuantityOne software (Biorad). B. OLFM4 overexpressed in COS7 cells, was added at approximately 3 ng/ml to oxaliplatin and sn38 treatment in the wild type HCT1 16 cells for 10 days. The colonies were stained and count as above. The histograms shown are representative of three individual experiments.
- the human colon adenocarcinoma cell line HCT1 16 (American Type Culture Collection, ATCC) were maintained in antibiotic-free RPMI 1640 medium (Lonza). Cultures were supplemented with 10 % fetal bovine serum. Cell lines were maintained at 37°C in 5 % C02 and were tested to rule out mycoplasma contamination.
- the plasmid pCMV/OLFM4 (Imagenes) was stably co-transfected with the pcDNA6/TR using LipofectAMINE 2000 reagent (Invitrogen) according to the manufacturer's instruction.
- HCT1 16 stable cells lines were selected with 100 ⁇ . ⁇ . " blasticidin (Sigma Aldrich, Deisenhofen, Germany) and 800 ⁇ . ⁇ 1 G418 for 1 month and maintained in RPMI 1640 medium supplement with 10% fetal bovine serum containing 5 ⁇ g.m 1 blasticidin and 200 ⁇ g.m 1 G418 OLFM4 expression was induced by 50 ng.rml "1 doxycycline for 48 h.
- the expression of OLFM4 protein in these stable cell lines was confirmed by Western blot as previously described.
- HCT1 16 stable OLFM4 cells were seeded at 300 cells into 6 well cell culture plates with RPMI 1640 medium supplement with 3% fetal bovine serum and incubated at 37°C in a 5% C0 2 atmosphere. Cells were then induced with 50 ng. mL "1 doxycycline (Sigma) for 24 h. After, the cells were traited with SN38 and Oxaliplatine and incubated at 37°C in a 5% C0 2 atmosphere for 10 days, washed twice with PBS and stained with 0,1 % crystal violet. The number of colonies exceeding 50 cells was visualized with a Bio-Rad Chemi Doc XRS Imaging device and counted using Quantity One imaging software (Bio-Rad). The survival fraction was determined as the ratio of the number of colonies observed without doxycycline to the number of cells with doxycycline, adjusted to the plating efficiency.
- Frozen sections (12 ⁇ thick) of either colon cancer or normal colonic mucosa were cut on a cryostat (Bright instrument Co Ltd, St Margarets Way, UK). The sections were then stained with toluidine blue using a rapid staining method. Toluidine blue stained sections were also prepared for visual reference. The tissue sections were put in the 70% ethanol bath for 1 minute, 95% ethanol for 2 minutes, 95% ethanol for 2 minutes, and finally, twice bath in 100% xylene for 5 minutes. Xylene was allowed to evaporate completely from the sections and then the sections were microdissected using a PixCell II laser capture microdissection system (Arcturus Engineering, Mountain View, California, USA).
- the laser capture system was equipped with PixCell II image archiving software (Arcturus Engineering). The settings of the laser were as follows: spot diameter set at 7.5 ⁇ , pulse duration 70 milliseconds, and power 70 mW. After microdissection, the plastic film containing the microdissected cells was removed and all the films containing material from a single sample placed in a microcentrifuge tube DNA extraction and protein lysis solution added. Approximately 30,000 cells were captured from a single or consecutive tissue sections using up to 5 CapSure LCM Caps (Molecular Devices Corporation), which were transferred to a 0.5 ml sterile Eppendorf tube for protein extraction (see below).
- Genomic DNA from frozen tissue was extracted with the PicoPureTM DNA extraction kit (Arturus Bioscience, Inc. Mountain View, CA, USA) and a whole genome amplification of genomic DNA was performed by polymerase chain reaction (PCR) using random 15-mer primers.
- PicoPureTM DNA extraction kit (Arturus Bioscience, Inc. Mountain View, CA, USA)
- PCR polymerase chain reaction
- Biotinylated PCR products were performed with an initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 30 s, primer annealing at 54°C for 30 s, and extension at 72°C for 1 min, followed by a final extension for 5 min at 72°C. All amplification reactions were performed in a DNAThermal Cycler 480 (Perkin Elmer, Boston, USA) with 1 unit of Taq Polymerase (Euroblue Taq-Eurobio, Les Ulis, France).
- the different sets of primers used to amplify the sequences of interest were: - PCR primer sequences by KRAS mutation for codons 12-13 (sense) 5'-AAC CTT ATG TGT GAC ATG TTC T-3' (SEQ ID NO. 1 ), (antisense): 5'-biotin-TCG TCC ACA AAA TGA TTC TGA-3' (SEQ ID NO. 2) and Sense Sequencing primer: 5'-CTT GTG GTA GTT GGA GC-3' (SEQ ID NO. 3) Codon 61 : primer sequence: (sense): 5'-TTA TGG CAA ATA CAC AAA GAA AGC-3' (SEQ ID NO.
- ssDNA was prepared from 40 ⁇ biotinylated PCR product using streptavidin-coated sepharose, and 1 .5 pmol of the sequencing primer was used for analysis. Sequencing was performed with the SNP Reagent Kit (Biotage) according to the manufacturer's instructions.
- Protein extraction was carried out using the Liquid Tissue MS Protein Prep kit according to manufacturer's protocol (Expression Pathology Inc., Gaithersburg, MD, USA). Briefly, the films from the underside of the caps for each sample were removed, transferred to low binding reaction tubes, and incubated with 20 ⁇ of Liquid Tissue extraction and heated at 95°C for 90 minutes. After cooling for 2 minutes on ice, 5 ⁇ of trypsin reagent was added and incubated at 37°C for one hour with vigorous shaking for 30 second at 20 minute intervals. Samples were further incubated overnight at 37°C followed by heating at 95°C for 5 minutes.
- the samples were harvested via centrifugation at 10,000 g, dried completely using a Speed-Vac and re-suspended in 100 ⁇ of 0.5% trifluoroacetic acid (TFA) in 5% acetonitrile, and were desalted via PepClean C-18 spin columns (Pierce Biotechnology, Rockford, IL) and dried for iTRAQTM processing.
- TFA trifluoroacetic acid
- Peptides samples were resuspended with 30 ⁇ of iTRAQ dissolution buffer (Applied Biosystems) and were reduced with 5 mM Tris-(2-carboxyethyl)phosphine (TCEP) at 60°C for 1 h and the cysteine-groups were blocked using a 10 mM methyl methanethiosulfonate (MMTS) solution at room temperature for 10 min.
- TCEP Tris-(2-carboxyethyl)phosphine
- MMTS methyl methanethiosulfonate
- Each peptide solution was labeled at room temperature for 2h with one iTRAQ reagent vial (mass tag 1 14, 1 15, 1 16 or 1 17) previously reconstituted with 70 ⁇ of ethanol.
- Peptide OFFGEL fractionation For pl-based peptide separation, we used the 3100 OFFGEL Fractionator (Agilent Technologies, Boblingen, Germany) with a 24-well set-up. Prior to electrofocusing, samples were desalted onto a Sep-Pak C18 cartridge (Waters). For 24-well set-up, peptide samples were diluted to a final volume of respectively 3.6 mL using OFFGEL peptide sample solution. To start, the IPG gel strips of 24 cm-long (GE Healthcare, Mijnchen, Germany) with a 3-10 linear pH range was rehydrated with the Peptide IPG Strip Rehydradation Solution according to the protocol of the manufacturer for 15 min. Then, 150 ⁇ of sample was loaded in each well.
- Electrofocusing of the peptides is performed at 20°C and 50 ⁇ until the 50 kVh level was reached.
- the 24 peptide fractions were withdrawn and the wells rinsed with 200 ⁇ _ of a solution of water/methanol/formic acid (49/50/1 ); after 15 min, the rinsing solutions were pooled with their corresponding peptide fraction. All fractions were evaporated by centrifugation under vacuum and maintained at -20°C. Just prior nano-LC, the fractions were resuspended in 20 ⁇ _ of H20 with 0.1 % (v/v) TFA.
- the samples were separated on an Ultimate 3,000 nano-LC system (Dionex, Sunnyvale, USA) using a C18 column (PepMap100, 3 ⁇ - ⁇ , 100A, 75 ⁇ id x 15cm, Dionex) at a flow rate of 300nl_/min.
- Buffer A was 2% ACN in water with 0.05% TFA and buffer B was 80% ACN in water with 0.04% TFA.
- Peptides were desalted for 3 min. using only buffer A on the precolumn, followed by a separation for 105 min. using the following gradient: 0 to 20% B in 10 min, 20% to 45% B in 85 min and 45% to 100% B in 10 min. Chromatograms were recorded at the wavelength of 214 nm.
- Peptide fractions were collected using a Probot microfraction collector (Dionex).
- CHCA LaserBioLabs, Sophia-Antipolis, France
- the matrix (concentration of 2mg/ml_ in 70% ACN in water with 0.1 % TFA) was continuously added to the column effluent via a micro "T" mixing piece at 1 .2 ⁇ _ ⁇ flow rate. After 12 min run, a start signal was sent to the Probot to initiate fractionation. Fractions were collected for 10 s and spotted on a MALDI sample plate (1 ,664 spots per plate, Applied Biosystems, Foster City, CA).
- MS and MS/MS analyses of off-line spotted peptide samples were performed using the 4800 MALDI-TOF/TOF Analyser (Applied Biosystems). After screening of all LC- MALDI sample positions in MS-positive reflector mode using 1500 laser shots, the fragmentation of automatically-selected precursors was performed at collision energy of 1 kV using air as collision gas (pressure of ⁇ 2 x 10-6 Torr). MS spectra were acquired between m/z 800 and 4000. For internal calibration, we used the parent ion of Glu1 -fibrinopeptide at m/z 1570.677 diluted in the matrix (3 femtomoles per spot).
- the software infers treatment-dependent changes in expression using Bayesian statistical methods. Basically, this approach was used to generate means, medians, and 95% credible intervals (upper and lower) for each treatment-dependent change in protein expression by using peptide-level data for each component peptide, and integrating data across the two experiments. For proteins whose iTRAQ ratios were downregulated in tissues, the extent of down- regulation was considered further if the null value of 1 was above the upper limit of the credible interval. Conversely, for proteins whose iTRAQ ratios were up-regulated in tumors, the extent of upregulation was considered further if the lower limit of the credible interval had a value greater than 1.
- the width of these credible intervals depends on the data available for a given protein. Since the number of peptides observed and the number of spectra used to quantify the change in expression for a given protein are taken into consideration, it is possible to detect small but significant changes in up- or downregulation when many peptides are available.
- Whole cell lysates were prepared from three normal tissues, three cancerous tissues non-mutated of KRAS gene and three cancerous tissues mutated of KRAS gene. Frozen tissue samples were homogenized and lysed in a buffer containing 7M urea, 2M thiourea and 4% (w/v) CHAPS at 4°C for 1 h using a rotary shaker. Lysis was achieved by sonication on ice (3 x 5 s pulses), and the lysates were clarified by centrifugation at 14,000 x g at 4°C for 15 min.
- Membranes were incubated with the respective secondary antibody, horseradish peroxidase-conjugated rabbit anti-lgG (goat anti-rabbit IgG, 1 :5000, Santa Cruz Biotechnology Inc.), and diluted with 1 % bovine serum albumin for 1 h at room temperature. After each step, blots were washed three times with 0.05% Tween, TBS. The membrane was probed with the indicated antibodies and developed with the ECL.
- a quantitative score was performed by estimating the percentage of immunopositive stained cells: 0, -10% cells; 1 , 10-30% cells; 2, 30-50% cells; 3, 50-70% cells; and 4, >70% cells.
- the intensity of staining was scored by evaluating the average staining intensity of the positive cells and secretion (0, none; 1 , weak; 2, intermediate; and 3, strong).
- the immunohistochemical data were subjected to statistical analysis as described above for the MS results.
- the purified culture media and lysate of COS-7 cells were pre-denatured in glycoprotein denaturing buffer at 100°C for 5 min.
- the denatured proteins were then treated with cocktail enzymes (Sigma-Aldrich Corporation) at 37°C for 3 h according to the manufacturer's recommendation, separated on 6% SDS-PAGE and followed by Western blot analysis.
- cocktail enzymes Sigma-Aldrich Corporation
- the pCMV-OLFM4 construct was transcribed and translated in the presence.
- Chromatin Immunoprecipitation Assays Cells, grown to 60% confluence, were treated or not as indicated and then washed and cross-linked with 1 % formaldehyde at room temperature for 8 min essentially as previously described (29, 30).
- Reaction was stopped with 10 ml of 125 mM glycin solution.
- Cells were washed with cold PBS and lysed in 500 ⁇ of lysis buffer (1 % SDS, 10 mM EDTA, 50 mM Tris-HCI pH 8.1 , 1 mM PMSF, 5 mM NaF, 5 mM Na3V04, 2 ⁇ g ml leupeptin, 5 ⁇ g ml aprotinin, 1 ⁇ g ml pestatin), and sonicated five times for 20 seconds each.
- lysis buffer (1 % SDS, 10 mM EDTA, 50 mM Tris-HCI pH 8.1 , 1 mM PMSF, 5 mM NaF, 5 mM Na3V04, 2 ⁇ g ml leupeptin, 5 ⁇ g ml aprotinin, 1 ⁇ g ml pestatin
- Immunoprecipitation was performed overnight with specific antibodies and IgG control, and then 2 ⁇ g of sheared salmon-sperm DNA and 20 ⁇ of protein G-agarose coated with salmon sperm DNA (Millipore) (of 50% slurry) were further added for 1 h at 4 °C. Note that immunoprecipitations were performed in the presence of 1 % Igepal CA-630.
- TSE I 0.1 % SDS, 1 % Triton X-100, 2 mM EDTA, 20 mM Tris-HCI pH 8.1 , 150 mM NaCI
- TSE II 0.1 % SDS, 1 % Triton X-100, 2 mM EDTA, 20 mM Tris-HCI pH 8.1 , 500 mM NaCI
- TP3 250 mM LiCI, 1 % NP-40, 1 % deoxycholate, 1 mM EDTA, 10 mM Tris-HCI pH 8.1 ).
- SEQ ID NO. 1 1 For 5'-TTCGCCGAGAAATCGTGGCTCT-3' SEQ ID NO. 12: Rev 5'- AG CTG AAC CACAG AC GGTTTG CT-3 ' RPLPO:
- SEQ ID NO. 13 For 5'-AACCCAGCTCTGGAGAAACT-3' SEQ ID NO. 14: Rev 5'-CCCCTGGAGATTTTAGTGGT-3' Other assays:
- the proportion of regulatory proteins such as kinases and transcription factors was determined.
- a total of 124 kinases were identified. This corresponds to 4.1 % of the 2971 proteins in our colorectal tumor proteome with GO annotations which is equivalent to the proportion expected from the human genome (4.1 %).
- GO cellular component analysis was performed and proteins were identified in several subcellular compartments such as mitochondrion, endoplasmic reticulum, nuclear part, cytosol, actin cytoskeleton, cytoplasmic vesicle, extracellular region and plasma membrane. All categories were overexpressed with respect to the genome, suggesting that our analysis detected preferentially abundant proteins
- HLA-DRA HLA-DRA (iTRAQ ratio 0.54) is one of the HLA class II alpha chain paralogues. It plays a central role in the immune system by presenting antigens derived from extracellular proteins. HLA-DRA is an inflammatory gene which has a role in colon carcinogenesis. Our result is consistent with previous study on HLA-DRA mRNA quantification which showed that HLA-DRA mRNA expression was significantly decreased in colorectal tumors from several cohorts. In our case, it should be noted that HLD-DRA expression is only significantly down-regulated in K-ras mutated tumors.
- GW1 12 also known as olfactomedin 4 (OLFM4) or human G-CSF clone-1 (hGC-1 ), was originally cloned from human myeloid cells and encodes a secreted glycoprotein of 510 amino acids.
- GW1 12 is normally expressed in the bone marrow, intestine and prostate, and altered expression is observed in various tumors including colon, breast, and lung cancers. Recent studies have suggested that GW1 12 is involved in the regulation of apoptosis. When overexpressed, this protein enhanced the survival of tumor cell and favored tumor growth.
- the OLFM4 gene is regulated by the N F-KB transcription factor to promote cell survival in gastric cancer cell.
- OLFM4 was shown to be a potential gastric cancer biomarker with 25% sensitivity for stage I, 63% for stage II, 40% for stage III and 30% for stage IV with a specificity of 95%. In our experimental conditions, OLFM4 was detected with the highest statistical confidence and we focused on this protein for the following part of the study. Validation of OLFM4 overexpression by immunohistochemistrv
- OLFM4 is a highly glycosylated secreted protein
- OLFM4 is secreted into extracellular medium
- a vector expressing the OLFM4 cDNA was transiently transfected into COS-7 cells.
- Culture media and cell lysates were collected 72 hr after transfection, and analyzed by Western blot.
- the resulting data showed that OLFM4 was detected in both culture medium and cell lysate (Fig. 3B).
- Fig. 3B This result further suggests that OLFM4 is exported into the extracellular medium as a secreted protein. Note that only the 72 kDa form was detected in the supernatant, indicating that this protein is secreted as a glycosylated protein.
- the western blot assay (Fig. 3A) suggested that the glycosylated olfactomedin-4, expressed as a 72 kDa isoform, was specific of K-ras mutated tumors and was not present in non-mutated tumors.
- we selected 43 independent tumors 26 WT and 17 KRAS
- 6 healthy controls for a second western blot assay.
- We calculated for each tissue the ratio between the 72 kDa and 54 kDa bands, which illustrates the ratio between the secreted form and the intracellular olfactomedin-4.
- the band at 72 kDa is always present, thus explaining the basal rate on the olfactomedin-4 in human serum; nevertheless, the ratio 72/55 always indicated a higher expression of the intracellular olfactomedin-4 with a median value of 0.72.
- the median was at 1 .27 and not significantly higher than that in healthy control.
- the Ras/NF-KB2 pathway promotes OLFM4 expression
- N F-KB transcription factor regulates OLFM4 expression in myeloid and gastric cells.
- a transcription recognition site analysis of the OLFM4 promoter showed the presence of different potential N F-KB2 binding sites (Fig 4C).
- N F-KB2 regulates OLFM4 expression in KRAS-mutated tumor
- Fig 4A we analyzed its activation following rasV12 induction in our colorectal model.
- Fig 4A The N F-KB2 activation is correlated with the Ras-mediated OLFM4 expression in colorectal cell lines.
- a downregulation of N F-KB2 inhibits OLFM4 expression at the protein and RNA level (Fig 4D-E).
- the secreted OLFM4 promotes resistance to SN38 and Oxaliplatin
- HCT1 16 stable cell lines that contain an inducible vector expressing the OLFM4 cDNA. Clonogenic assays performed on these cells indicate that OLFM4 increased cell survival in response to oxaliplatin and sn38 as compared to wild type HCT1 16 cells (Fig. 5A and C). Despite changes in oxaliplatin and sn38 sensitivity, the morphology (data not shown) and growth rate of the HCT1 16-OLFM4 and HCT1 16-WT were similar.
- HCT1 16 cells were treated with chemotherapeutic drugs in the presence or absence of OLFM4.
- chemotherapeutic drugs in the presence or absence of OLFM4.
- clonogenic assay we have shown that the presence of OLFM4 in the media promotes resistance to the genotoxic agents, oxaliplatin and sn38, the active metabolite of irinotecan (Fig. 5B and D). All differences were shown to be significant by statistical analysis. These results suggest that extracellular OLFM4 induces resistance to sn38 and oxaliplatin in colorectal cells.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biomedical Technology (AREA)
- Oncology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Hospice & Palliative Care (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Cell Biology (AREA)
- Mycology (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- Inorganic Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
Claims
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/IB2011/000787 WO2012117267A1 (en) | 2011-03-02 | 2011-03-02 | Use of the olfactomedin-4 protein (olfm4) in colorectal cancer diagnosis |
Publications (2)
Publication Number | Publication Date |
---|---|
EP2681330A1 true EP2681330A1 (en) | 2014-01-08 |
EP2681330B1 EP2681330B1 (en) | 2017-12-27 |
Family
ID=44626368
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP11719644.4A Not-in-force EP2681330B1 (en) | 2011-03-02 | 2011-03-02 | Use of the olfactomedin-4 protein (olfm4) in colorectal cancer diagnosis |
Country Status (5)
Country | Link |
---|---|
US (1) | US9523691B2 (en) |
EP (1) | EP2681330B1 (en) |
CN (1) | CN103502470B (en) |
ES (1) | ES2663843T3 (en) |
WO (1) | WO2012117267A1 (en) |
Families Citing this family (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2876442A1 (en) * | 2013-11-22 | 2015-05-27 | Institut de Cancérologie de l'Ouest | Olfactomedin-4, neudesin and desmoplakin as biomarkers of breast cancer |
FR3017713B1 (en) * | 2014-02-18 | 2018-01-12 | Biomerieux | METHOD AND KIT FOR DETERMINING THE PROBABILITY FOR A PATIENT TO EVOLVE TO A SEVERE DENGUE |
US10407737B2 (en) * | 2014-04-10 | 2019-09-10 | Bio-Marcare Technologies Ltd. | Methods and kits for identifying pre-cancerous colorectal polyps and colorectal cancer |
US20160209415A1 (en) * | 2015-01-20 | 2016-07-21 | Poochon Scientific LLC | Method to predict or diagnose a colorectal cancer |
WO2017129537A1 (en) * | 2016-01-25 | 2017-08-03 | Sanofi | Method for predicting the outcome of a treatment with aflibercept of a patient suspected to suffer from a cancer by measuring the level of a plasma biomarker |
CN105779635B (en) * | 2016-05-18 | 2019-10-22 | 黄文林 | A kind of kit being used for colorectal cancer Index for diagnosis based on OLFM1 gene |
US20190285635A1 (en) * | 2016-12-09 | 2019-09-19 | Shenzhen Senboll Biotechnology Co., Ltd. | Cnpy2 isoform 2 and the use thereof as molecular marker for colorectal cancer |
CN111621561B (en) * | 2020-06-15 | 2021-12-21 | 浙江大学 | Application of OLFM4 in nonalcoholic fatty liver disease (NAFLD) |
CN115786518A (en) * | 2022-11-29 | 2023-03-14 | 广东医科大学附属医院 | Application of olfactin structural domain family protein 4 (OLFM 4) in colon cancer detection product |
-
2011
- 2011-03-02 US US14/002,225 patent/US9523691B2/en active Active
- 2011-03-02 ES ES11719644.4T patent/ES2663843T3/en active Active
- 2011-03-02 CN CN201180070187.7A patent/CN103502470B/en not_active Expired - Fee Related
- 2011-03-02 WO PCT/IB2011/000787 patent/WO2012117267A1/en active Application Filing
- 2011-03-02 EP EP11719644.4A patent/EP2681330B1/en not_active Not-in-force
Also Published As
Publication number | Publication date |
---|---|
US20130336969A1 (en) | 2013-12-19 |
CN103502470B (en) | 2016-12-07 |
WO2012117267A1 (en) | 2012-09-07 |
EP2681330B1 (en) | 2017-12-27 |
ES2663843T3 (en) | 2018-04-17 |
CN103502470A (en) | 2014-01-08 |
US9523691B2 (en) | 2016-12-20 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP2681330B1 (en) | Use of the olfactomedin-4 protein (olfm4) in colorectal cancer diagnosis | |
EP1751309B1 (en) | Methods for prediction of clinical outcome to epidermal growth factor receptor inhibitors by cancer patients | |
JP5926487B2 (en) | Method for treating cancer resistant to ErbB therapy | |
JP5671206B2 (en) | Mutation of c-KIT oncogene in melanoma | |
US20130230511A1 (en) | Biomarkers for response to tyrosine kinase pathway inhibitors in cancer | |
KR20140132712A (en) | Cancer patient selection for administration of wnt signaling inhibitors using rnf43 mutation status | |
JP2020040959A (en) | Use of egfr biomarkers for treatment of gastric cancer with anti-egfr agents | |
US20140030257A1 (en) | Agtr1 as a marker for bevacizumab combination therapies | |
US8216783B2 (en) | Over-expression and mutation of a tyrosine kinase receptor FGFR4 in tumors | |
JP6858563B2 (en) | Prediction of EGFR inhibitor effect by BRAF mutation detection | |
JP5923450B2 (en) | MITF as a marker for predisposition to cancer | |
EP3515418B1 (en) | Use of c-met inhibitors to treat cancers harbouring met mutations | |
US9028831B2 (en) | Markers for selecting personalized therapies for the treatment of cancer | |
EP2492688A1 (en) | Molecular biomarkers for predicting response to antitumor treatment in lung cancer | |
US20120316187A1 (en) | Molecular biomarkers for predicting response to tyrosine kinase inhibitors in lung cancer | |
WO2010054045A1 (en) | Method for optimizing the treatment of chronic myeloid leukemia with abl tyrosine kinase inhibitors | |
KR20160003646A (en) | Use of egfr biomarkers for the treatment of gastric cancer with anti-egfr agents |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 20131002 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR |
|
DAX | Request for extension of the european patent (deleted) | ||
17Q | First examination report despatched |
Effective date: 20161125 |
|
GRAP | Despatch of communication of intention to grant a patent |
Free format text: ORIGINAL CODE: EPIDOSNIGR1 |
|
INTG | Intention to grant announced |
Effective date: 20170717 |
|
GRAS | Grant fee paid |
Free format text: ORIGINAL CODE: EPIDOSNIGR3 |
|
GRAA | (expected) grant |
Free format text: ORIGINAL CODE: 0009210 |
|
AK | Designated contracting states |
Kind code of ref document: B1 Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR |
|
REG | Reference to a national code |
Ref country code: GB Ref legal event code: FG4D |
|
REG | Reference to a national code |
Ref country code: CH Ref legal event code: EP |
|
REG | Reference to a national code |
Ref country code: AT Ref legal event code: REF Ref document number: 958344 Country of ref document: AT Kind code of ref document: T Effective date: 20180115 |
|
REG | Reference to a national code |
Ref country code: IE Ref legal event code: FG4D |
|
REG | Reference to a national code |
Ref country code: DE Ref legal event code: R096 Ref document number: 602011044532 Country of ref document: DE |
|
REG | Reference to a national code |
Ref country code: FR Ref legal event code: PLFP Year of fee payment: 8 |
|
REG | Reference to a national code |
Ref country code: CH Ref legal event code: NV Representative=s name: MICHELI AND CIE SA, CH |
|
REG | Reference to a national code |
Ref country code: NL Ref legal event code: FP |
|
REG | Reference to a national code |
Ref country code: ES Ref legal event code: FG2A Ref document number: 2663843 Country of ref document: ES Kind code of ref document: T3 Effective date: 20180417 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: NO Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20180327 Ref country code: LT Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20171227 Ref country code: FI Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20171227 |
|
REG | Reference to a national code |
Ref country code: LT Ref legal event code: MG4D |
|
REG | Reference to a national code |
Ref country code: AT Ref legal event code: MK05 Ref document number: 958344 Country of ref document: AT Kind code of ref document: T Effective date: 20171227 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: LV Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20171227 Ref country code: HR Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20171227 Ref country code: BG Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20180327 Ref country code: GR Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20180328 Ref country code: RS Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20171227 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: EE Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20171227 Ref country code: CY Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20171227 Ref country code: CZ Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20171227 Ref country code: SK Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20171227 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: SM Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20171227 Ref country code: AT Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20171227 Ref country code: IS Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20180427 Ref country code: RO Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20171227 Ref country code: PL Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20171227 |
|
REG | Reference to a national code |
Ref country code: DE Ref legal event code: R097 Ref document number: 602011044532 Country of ref document: DE |
|
PLBE | No opposition filed within time limit |
Free format text: ORIGINAL CODE: 0009261 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: DK Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20171227 Ref country code: MC Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20171227 |
|
26N | No opposition filed |
Effective date: 20180928 |
|
REG | Reference to a national code |
Ref country code: IE Ref legal event code: MM4A |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: LU Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20180302 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: IE Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20180302 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: SI Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20171227 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: MT Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20180302 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: TR Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20171227 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: HU Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT; INVALID AB INITIO Effective date: 20110302 Ref country code: PT Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20171227 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: SE Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20171227 Ref country code: MK Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20171227 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: AL Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20171227 |
|
PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: GB Payment date: 20220316 Year of fee payment: 12 Ref country code: DE Payment date: 20220307 Year of fee payment: 12 Ref country code: CH Payment date: 20220321 Year of fee payment: 12 |
|
PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: NL Payment date: 20220225 Year of fee payment: 12 Ref country code: IT Payment date: 20220309 Year of fee payment: 12 Ref country code: FR Payment date: 20220331 Year of fee payment: 12 Ref country code: BE Payment date: 20220316 Year of fee payment: 12 |
|
PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: ES Payment date: 20220404 Year of fee payment: 12 |
|
REG | Reference to a national code |
Ref country code: DE Ref legal event code: R119 Ref document number: 602011044532 Country of ref document: DE |
|
REG | Reference to a national code |
Ref country code: CH Ref legal event code: PL |
|
REG | Reference to a national code |
Ref country code: NL Ref legal event code: MM Effective date: 20230401 |
|
GBPC | Gb: european patent ceased through non-payment of renewal fee |
Effective date: 20230302 |
|
REG | Reference to a national code |
Ref country code: BE Ref legal event code: MM Effective date: 20230331 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: NL Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20230401 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: GB Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20230302 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: LI Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20230331 Ref country code: GB Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20230302 Ref country code: FR Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20230331 Ref country code: DE Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20231003 Ref country code: CH Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20230331 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: BE Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20230331 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: ES Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20230303 |
|
REG | Reference to a national code |
Ref country code: ES Ref legal event code: FD2A Effective date: 20240426 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: IT Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20230302 Ref country code: ES Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20230303 |